Goodpasture Antigen Binding Protein and its Detection

ABSTRACT

The present invention provides native Goodpasture antigen binding protein isoforms, monoclonal antibodies directed against such proteins, and methods for their use.

CROSS REFERENCE

This application is a divisional of U.S. application Ser. No. 12/506,064filed Jul. 20, 2009, which claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 61/082,741 filed Jul. 22, 2008 and 61/085,211filed Jul. 31, 2008, all of which are incorporated by reference hereinin their entirety.

BACKGROUND OF THE INVENTION

The conformation of the non-collagenous (NC1) domain of the α3 chain ofthe basement membrane collagen IV [α3(IV)NC1] depends in part onphosphorylation. Goodpasture Antigen Binding Protein (GPBP) (WO00/50607; WO 02/061430) is a novel non-conventional protein kinase thatcatalyzes the conformational isomerization of the α3(IV)NC1 domainduring its supramolecular assembly, resulting in the production andstabilization of multiple α3(IV)NC1 conformers in basement membranes.Elevated levels of GPBP have been associated with the production ofnon-tolerized α3(IV)NC1 conformers, which conduct the autoimmuneresponse mediating Goodpasture (“GP”) disease. In GP patients,autoantibodies against the non-collagenous C-terminal domain (NC1) ofthe type IV collagen α3 chain (“Goodpasture antigen” or “GP antigen”)cause a rapidly progressive glomerulonephritis and often lunghemorrhage, the two cardinal clinical manifestations of the GP syndrome.

The identification of GPBP provided methods for identification ofcompounds for the treatment of autoimmune disorders, cancer, proteinmisfolding-mediated disorders and aberrant apoptosis, and also providedpotential therapeutics for these disorders. Thus, the identification ofnovel GPBP isoforms would be advantageous in at least these fields.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides isolated polypeptidesof 90% or greater purity consisting of the amino acid sequence of SEQ IDNO: 2 (91 kD GPBP).

In a second aspect, the present invention provides substantiallypurified recombinant polypeptides comprising the general formula X-SEQID NO:2, wherein X is a detectable polypeptide. In one preferredembodiment of this aspect, the detectable polypeptide is selected fromthe group consisting of fluorescent polypeptides and polypeptide membersof a binding pair. In another aspect, the present invention providessubstantially purified nucleic acids encoding the polypeptides of thissecond aspect of the invention.

In a third aspect, the present invention provides substantially purifiednucleic acids encoding a polypeptide consisting of the amino acidsequence of SEQ ID NO:2 (91 kD GPBP). In one preferred embodiment, thesubstantially purified nucleic acids consist of the nucleic acid of SEQID NO:1, or a mRNA product thereof.

In a fourth aspect, the present invention provides recombinantexpression vectors comprising the substantially purified nucleic acid ofany aspect of the invention.

In a fifth aspect, the present invention provides host cells transfectedwith a recombinant expression vector of the invention.

In a sixth aspect, the present invention provides a substantiallypurified polypeptide comprising the amino acid sequence of SEQ ID NO:2(91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP), wherein the polypeptide of SEQID NO:2 or SEQ ID NO:4 comprises one or more post-translationalmodifications (PTMs) directly and/or indirectly involving amino acidsresidues 305-344 GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO:10) (numbering based on position within 77 kD GPBP). In one preferredembodiment, the one or more PTMs comprise covalent PTMs. In anotherpreferred embodiment, the one or more PTMs comprise covalent PTMs withinamino acids 305-344 (SEQ ID NO: 10). In one preferred embodiment the oneor more PTMs directly or indirectly involve residues 320-327 (EEFFDAVE,SEQ ID NO:5). In another preferred embodiment, the one or more PTMscomprise one or more covalent PTMs within residues 320-327 (EEFFDAVE,SEQ ID NO:5). In various preferred embodiments of this aspect, thesubstantially purified polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP).

In a seventh aspect, the present invention provides substantiallypurified polypeptides comprising the amino acid sequence of SEQ ID NO:2(91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP), wherein the polypeptide of SEQID NO:2 or SEQ ID NO:4 comprises one or more PTMs directly and/orindirectly involving residues 371-396, PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQID NO:9) (numbering based on positions within 77 kD GPBP). In onepreferred embodiment, the one or more PTMs comprise covalent PTMs. Inanother preferred embodiment, the one or more PTMs comprise covalentPTMs within amino acids 371-396 (SEQ ID NO:9). In one preferredembodiment, the one or more PTMs directly or indirectly involve residues388-392 (DDVHR, SEQ ID NO:6). In another preferred embodiment, the oneor more PTMs comprise one or more covalent PTMs within residues 388-392(SEQ ID NO:6) In another preferred embodiment, the polypeptide furthercomprises one or more PTMs directly and/or indirectly involving aminoacids residues 305-344 GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ IDNO: 10) (numbering based on position within 77 kD GPBP); preferably theone or more PTMs comprise covalent PTMs, and even more preferably theone or more PTMs comprise covalent PTMs within amino acids 305-344 (SEQID NO: 10). In another preferred embodiment the one or more PTMsdirectly or indirectly involve residues 320-327 (EEFFDAVE, SEQ ID NO:5).In another preferred embodiment, the one or more PTMs comprise one ormore covalent PTMs within residues 320-327 (EEFFDAVE, SEQ ID NO:5). Invarious preferred embodiments of this aspect, the substantially purifiedpolypeptide comprises or consists of the amino acid sequence of SEQ IDNO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP).

In an eighth aspect, the present invention provides substantiallypurified monoclonal antibodies that selectively bind to a polypeptide ofthe sixth or seventh aspect of the invention.

In a ninth aspect, the present invention provides substantially purifiedmonoclonal antibodies that specifically binds to the polypeptide of SEQID NO:2 and not to the polypeptide of SEQ ID NO:4. In one preferredembodiment, the monoclonal antibody binds to an epitope within the aminoacid sequence DGWKGRLPSPLVLLPRSARC (SEQ ID NO:7)

In a tenth aspect, the present invention provides methods for detectingcirculating Goodpasture antigen binding protein (GPBP), comprising

(a) contacting a plasma sample with a GPBP-binding molecule underconditions to promote selective binding of the GPBP-binding molecule tothe GPBP;

(b) removing unbound GPBP-binding molecules; and

(c) detecting complex formation between GPBP-binding molecule and theGPBP in the plasma sample.

In an eleventh aspect, the present invention provides methods fordetecting urinary Goodpasture antigen binding protein (GPBP), comprising

(a) contacting a urine sample with a GPBP-binding molecule underconditions to promote selective binding of the GPBP-binding molecule tothe GPBP;

(b) removing unbound GPBP-binding molecules; and

(c) detecting complex formation between GPBP-binding molecule and theGPBP in the urine sample.

In a twelfth aspect, the present invention provides methods forisolating native 77 kD GPBP, comprising:

(a) subjecting a plasma sample to ammonium sulfate precipitation;

(b) conducting ion-exchange chromatography (IEC) on the ammonium sulfateprecipitated serum sample;

(c) identifying IEC fractions containing native 77 kD GPBP;

(d) subjecting IEC fractions containing native 77-GPBP to gel filtrationchromatography (GFC); and

(e) identifying GFC fractions containing native 77 kD GPBP.

In a thirteenth aspect, the present invention provides methods forisolating native 91 kD GPBP, comprising:

(a) subjecting a urine sample to salt precipitation;

(b) conducting double ion exchange chromatography (IEC) on the saltprecipitated protein sample; and

(c) identifying IEC fractions containing native 91 kD GPBP.

In a fourteenth aspect, the present invention provides methods forisolating native GPBP isoforms, comprising:

(a) passing a plasma sample or urine sample through an immunoaffinitycolumn containing GPBP-binding molecules that selectively bind to nativeGPBP;

(b) washing unbound protein from the plasma or urine sample from theimmunoaffinity column; and

(c) eluting native GPBP isoforms from the column.

In one preferred embodiment, these methods can be used, for example, tosubstantially purify native 77 kD GPBP and native 91 kD GPBP from plasmaand urine, respectively, as disclosed in more detail in the examplesthat follow. In another preferred embodiment, the GPBP-binding moleculescomprise GPBP antibodies. In another preferred embodiment, theantibodies comprise the novel monoclonal antibodies of the presentinvention. In another preferred embodiment, the eluting step comprisesuse of a denaturing eluting buffer.

DESCRIPTION OF THE FIGURES

FIG. 1. COL4A3BP encodes for polypeptides of 77-, 91- and 120-kDa. In A,FLAG-tagged GPBP or GPBPΔ26/CERT (10-20 ng) were analyzed by Westernblot with the indicated antibodies. In B, cell extracts (50 μg) wereanalyzed as in A. In C, extracts (10 μg) from control cells (−) or cellsexpressing pc-n4′ were analyzed as in A.

In D, extracts (50 μg) from untransfected cells (−) or from cellstransfected with the indicated siRNA-expressing plasmid were analyzed asin A. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as aloading control and siRNA specificity. The reactivity of mAb e26 withnative or recombinant polypeptides was fully abolished when usingGPBPpep1 (20 μM) as antibody blocking peptide (not shown). In this andfollowing Figures, numbers and bars or arrows indicate the size in kDaand the positions of the MW standards or the reactive polypeptides,respectively. The results shown in this and following Figures arerepresentative of at least two independent experiments.

FIG. 2. GPBP polypeptides of 91- and 120-kDa are the products of mRNAnoncanonical translation initiation. In A, schematic representation ofthe cDNAs used to construct the indicated plasmids. In B, cell extracts(10 μg) (ex vivo) or individual transcription/translation mixtures (invitro) expressing the indicated plasmid construct were analyzed byWestern blot using mAb e26 (ex vivo) or by fluorography (in vitro).Lysates from untransfected cells (ex vivo) or mixtures without plasmid(in vitro) were used as Control. In C, indicated are the sequence of theN terminal open reading frame (ORF) of GPBP in one-letter code (SEQ IDNO:15) and the corresponding mRNA nucleotide sequence (SEQ ID NO:14) incapital letters. The gray and black letters indicate the 5′-UTR and ATR,respectively. Boxed are the codons and residues for canonical andnoncanonical translation initiation. The peptide sequence targeted by Ab24 is highlighted in gray. The negative numbers at right denote theposition of the codon or residue from canonical translation initiationsite (AUG or Met, +1). In D, extracts (10 μg) from cells not expressing(Control) or expressing the indicated plasmid constructs without (−) orwith a stop codon at the indicated positions were analyzed by Westernblot using the indicated antibodies. In E, partially purified cellextracts (50 μg) were analyzed by Western blot using the indicatedreactive species and a non-reactive F(ab)₂ Ab 20 (Cont).

FIG. 3. The 91- and 120-kDa GPBP isoforms are insoluble membrane-boundpolypeptides. In A, intact cells were incubated with αGPBP-Alexa Fluor647 antibodies (αGPBP-AF647) in the presence of GPBPpep1 orequimolecular amount of a nonrelevant peptide (Contpep) and Rhodamine123 for mitochondrial staining of living cells, and analyzed by confocalmicroscopy. Scale bar, 21 μM. In B, cells were detached and incubatedwith blocking solution in the absence (control) or presence ofbiotinylated αGPBP antibodies (αGPBP). The cell surface-bound antibodywas detected with streptavidin-FITC and flow cytometry. As a control,parallel cultures were incubated with the same antibodies in thepresence of GPBPpep1 (αGPBP+GPBPpep1) or equimolecular amount of anon-relevant peptide (αGPBP+Contpep) and similarly analyzed. In C,similar amounts (10 μg) of the indicated cellular fractions wereanalyzed by Western blot using antibodies for the indicated proteins. Weused as cellular compartment markers: pyruvate dehydrogenase (PDH) formitochondria; cathepsine D for lysosome; prion protein (PrP) formicrosome; and nuclear factor kappa B (p65) for nucleus and cytosol. ForGPBP and GPBPΔ26/CERT detection, we used mAb e26 and mAb 14,respectively. Since we did not detect expression of 77-kDa GPBP in thecytosol, mAb 14 reactivity in this compartment can be attributed toGPBPΔ26/CERT.

FIG. 4. The 77-kDa GPBP isoform interacts with type IV collagen incultured cells. In A, HEK 293 or HEK 293-FLAG-α3(IV) cells werecross-linked, lysed and αFLAG extracted. Fifty micrograms of cell lysate(Input) or the corresponding FLAG-immunoprecipitated materials (IPαFLAG) were reversed cross-linked and analyzed by Coomassie bluestaining or Western blot with αGPBPr. The major specific polypeptides inFLAG-immunoprecipitates (arrows) were excised and identified byMALD/TOF/TOF mass spectrometry. In B, HEK 293 (−) or HEK 293-FLAG-α3(IV)(+) cells were transfected with pcDNA3 (−) or with pc-n4′ (+),cross-linked, processed and analyzed as in A by Western blot using theindicated antibodies.

FIG. 5. Export of 77-kDa GPBP to the extracellular compartment. In A,HeLa cells were transfected with the indicated plasmid constructs, andthe indicated proteins visualized by standard indirectimmunofluorescence. DNA was stained with 4′-6′-diamino-2-phenylindole(DAPI) for nuclear visualization. Original magnifications×400. In B andC, extracts (10 μg) from cells expressing the indicated plasmidconstructs (lysates) or FLAG-immunoprecipitates from the correspondingculture media (media IP) were analyzed by Western blot using theindicated antibodies.

FIG. 6. The 91-kDa GPBP regulates 77-kDa GPBP secretion in culturedcells. In A, extracts (10 μg) from two independent clones expressing(c8) or not expressing (c19) recombinant 91-kDa GPBP were analyzed byWestern blot with mAb 14 antibodies, which react poorly with native91-kDa counterpart (FIG. 1B). In B, the same clones were transfectedwith pc-FLAG-GPBP, and cell extracts (lysates) orFLAG-immunoprecipitates from the corresponding culture media (media IP)were analyzed by Western blot using the indicated antibodies. Similarconclusions were obtained when assaying c14, an independent HEK 293clone expressing levels of recombinant 91-kDa GPBP similar to c8 (notshown).

FIG. 7. GPBPΔ26/CERT but not GPBP is sensitive to sphingomyelinase celltreatment. In A, HeLa cells were transfected with the indicated plasmidcontracts and treated (+) or not (−) with spingomyelinase, lysed,FLAG-immunoprecipitated, and analyzed by Western blot with αFLAGantibodies (bSMase) Immunoprecipitates from untreated cells wereincubated (+) or not (−) with phosphatase and similarly analyzed(λPPase). We have used a 8-12% gradient gel and extensiveelectrophoresis to separate phosphorylated and dephosphorylated versionsof GPBPΔ26/CERT and estimated their relative abundance by Western blotand densitometry. In B, the same cells as in A were fixed bymethanol/acetone, double-labeled with anti-FLAG-FITC antibody (green)and DAPI (blue) and analyzed by direct immunofluorescence. Originalmagnification×400.

FIG. 8. The levels of circulating 77-kDa GPBP are up-regulated inGoodpasture patients and in animal models of immune complex-mediatedglomerulonephritis. In A, material isolated by immunoaffinitychromatography from a Goodpasture patient plasmapheresis was analyzed byWestern blot in the presence (αGPBP) or absence (Control) ofGPBP-specific antibodies. In B, is the plot representing the standardcurve obtained from an ELISA performed as in Material and Methodsindicated using recombinant GPBP. In C and D are scatter plots ofintensity of fluorescence (I.F.) in arbitrary units (A.U.) measured bysimilar ELISA. Sera from healthy donors (Control), Goodpasture patients(GP), and from NZW mice of the indicated ages were diluted 1:10. Thefluorescence in the absence of sera was considered background andsubtracted from each individual value. In both series, P<0.001. Barsindicate the mean of each series and a circle represents the mean valueof individual samples. In A-D, αGPBPr was the capturing and αGPBPab thedetecting antibodies.

FIG. 9. The binding site of mAb 14 maps to the FFAT motif of GPBP. In A,indicated in one-letter code is the primary structure of the FFAT motifand flanking region in GPBP (residues 316-333) (SEQ ID NO:8) and thehomologous region in GPBP_(ΔFFAT) (SEQ ID NO:29) where dashes indicatethe deleted residues within FFAT motif (boxed). In B, cell extracts (10μg) expressing the indicated proteins were analyzed by Western blotusing the indicated antibodies.

FIG. 10. Recombinant GPBP expression induces accumulation of GPBPpolypeptides in the cytosol. Cells were transfected with the indicatedplasmid constructs, collected one day after transfection, subjected tofractionation as indicated in Material and Methods in the Example 1 andanalyzed by Western blot as in FIG. 3C using the indicated antibodies.Arrows and numbers indicate the position and M_(r) in kDa of thedifferent GPBP polypeptides. The 120-kDa polypeptide was mainly found inlysosomal fraction and in a more limited amounts in microsomal fraction,further suggesting that it represents a covalently modified-derivedversion of the 91-kDa generated in the secretory pathway. Additionalobservations include the comparatively lower reactivity that mAb e26displays towards the 91-kDa polypeptide that resides in the cytosol(compare mAb e26 with mAb 14 reactivity when the polypeptide resides incytosol or microsomes—150,000×g).

FIG. 11. Extracellular 77-kDa GPBP does not react significantly with mAbe26. Cells transfected with pc-FLAG-GPBP were lysed and thecorresponding cultured media subjected to FLAG-immunoprecipitation.Similar amounts of cell extracts (lysate) or immunoprecipitates (mediaIP) were analyzed by Western blot using the indicated antibodies.

FIG. 12: Western blot analysis of GPBP isolated from plasma samplesusing chemical techniques. The GPBP partially purified fromapproximately 1.25 mL of human plasma (see Example 2) was analyzed byWestern blot under reducing conditions using HRP-labeled mAb N 27.Arrows and numbers indicated the position and the estimated M_(r) forreactive polypeptides.

FIG. 13. GPBP isolated from urine of a control donor usingimmunoaffinity chromatography. Two hundred and fifty milliliters ofurine from a control donor (previously cleared by centrifugation andneutralized with Tris), were loaded onto a 1 mL column of Sepharose4B-conjugated with 200 μg of rabbit polyclonal anti-GPBP antibodies. Thecolumn was washed with 30 mL of TBS and the bound material was elutedwith Gentle Immunopure™ Elution Buffer (Pierce). The material eluted wasdialyzed against TBS and further analyzed by Western blot usingGPBP-specific chicken polyclonal antibodies (αGPBPch) and HRP-labelledanti-chicken IgY (secondary antibody). Antibody specificity wasconfirmed by staining a control lane loaded with the same material withsecondary antibody (Cont). Bars and numbers or arrows and numbersindicate the position and size (kDa) of MW standards (left) or GPBPpolypeptides (right), respectively.

FIG. 14. Indirect ELISA to detect GPBP in urine samples. RecombinantGPBP diluted in urine and urine samples from seven donors (1-7) werecoated onto ELISA plates overnight at 4° C. Plates were blocked with 3%BSA in PBS and immunodetection performed with GPBP-specific chickenpolyclonal antibodies (αGPBPch) and HRP-labelled anti-chicken IgY(secondary antibody). Amplex UltraRed reagent (Invitrogen) was used fordeveloping the plate. In A, is represented a scatter plot on a log-logscale of the indicated concentrations of GPBP versus fluorescenceintensity (F.I.) expressed in arbitrary units (A.U.). In B, isrepresented the linear regression line calculated with the indicatedconcentrations and their respective F.I. values plotted on linear scale,that was used to determine GPBP sample concentration in D. In C, isrepresented raw data obtained analyzing donor samples with: secondaryantibody (Cont), nonspecific chicken IgY and secondary antibody (IgY),or with αGPBPch and secondary antibody (αGPBPch). In D, the table showscorresponding transformed data using the curve obtained in B.

FIG. 15. Salting-out and ion exchange chromatography of urine samples.Four hundred milliliters of urine cleared by centrifugation was broughtto 0.85 M NaCl overnight at 4° C., and subjected to centrifugation at10.000×g for 30 min at 4° C. A sample of the supernatant (Spt 0.85 MNaCl) was stored at 4° C. to be included in the subsequent analysis. Theresulting pellet was dissolved in 50 mM Tris pH 7.5, dialyzed againstthe same buffer, extracted with 0.7 mL of CM resin and unbound materialfurther extracted with 0.5 mL of DEAE resin. CM resin was eluted with 1MNaCl, 50 mM Tris pH 7.5 (CM, 1M NaCl), and DEAE resin was subsequentlyeluted with 0.35M NaCl, 50 mM Tris pH 7.5 (DEAE, 0.35M NaCl) and 1MNaCl, 50 mM Tris pH 7.5 (DEAE, 1M NaCl). Equivalent amounts of eachsample including the supernatant of the DEAE extraction (Spt CM/DEAE)were analyzed by Western blot with GPBP-specific chicken polyclonalantibodies and HRP-labelled anti-chicken IgY (αGPBPch). Nonspecificreactive polypeptides were identified by staining an in-parallelanalysis using only HRP-labelled anti-chicken IgY (Cont). Bars andnumbers or arrows and numbers indicate the position and size (kDa) of MWstandards (left) or polypeptides specifically reacting with anti-GPBPantibodies and that were detected only in SptCM/DEAE (right),respectively.

FIG. 16. Western blot analysis of intracellular and extracellularFLAG-GPBP produced in HEK 293 cells using individual N1-N28 monoclonalantibodies. At the upper composite, 10 μg of total protein extract fromHEK 293 cells expressing recombinant FLAG-GPBP were subjected to Westernblot analysis using N1-N28 antibodies (1-28). A major polypeptide of˜77-kDa representing the full length recombinant GPBP polypeptide andvariable presence of derived polypeptides of lower M_(r) (45-77 kDa)were observed. At the lower composite, the same antibodies were assayedagainst extracellular recombinant GPBP (77-kDa polypeptide) purified byanti-FLAG immunoprecipitation from the culture media of FLAG-GPBPexpressing HEK293 cells (Revert et al. 2008 J. Biol. Chem.283:30246-55). A major polypeptide 77-kDa representing the full lengthFLAG-GPBP polypeptide was detected along with a minor nonspecificpolypeptide of lower M_(r) (Conj), which reacted with the secondaryantibody (anti-mouse IgG) and is suspected to represent derived productsfrom the immunoprecipitating antibody (mouse anti-FLAG IgG) (not shown).Unless otherwise indicated, in this and subsequent Western blots, 1-28is N1-N28, and anti-mouse-HRP and chemiluminescence were used fordeveloping purposes.

FIG. 17. Western blot analysis of HEK 293 cell extracts using N1-N27monoclonal antibodies. Fifty μg of HEK 293 cell extract were analysed byWestern blot using the indicated antibodies. The antibodies recognizedfour distinct GPBP-related polypeptides: the 77-kDa canonicalpolypeptide, a 45-kDa fragment, an 88-kDa band, and a 91-kDa polypeptidealso targeted by mAb e26. The polypeptide pinpointed by the arrow wasrecognized by the secondary antibody (anti-mouse IgG HRP-labelled) andtherefore does not represents a GPBP product. The origin of 88-kDapolypeptide is unknown although its M_(r) suggest that it represents aphosphorylated version of the 77-kDa canonical polypeptide.

FIG. 18. Cloning of GPBP deletion mutants. In A, on the primarystructure of GPBP (SEQ ID NO:4) we indicate the C terminus (bent arrows)of the thirteen 3′ terminal FLAG-GPBP cDNA deletion mutants (1-13),obtained by standard PCR and recombinant DNA procedures. In B, is shownthe sequence of GPBP encompassing the C-terminal regions of deletionmutants 7 (upper box) and 8 (lower box). In each lane, the number of thelast residue is indicated. Δ1 is a FLAG-GPBP deletion mutant lackingresidues 285-304 and similarly Δ2-Δ4 mutants lack residues 305-324,325-344 and 345-364, respectively (SEQ ID NOS: 30-33). A peptiderepresenting the bold sequence (SEQ ID NO:8) efficiently competed mAb 14binding to GPBP and a GPBP mutant containing the sequence Ala Ala Valinstead of the underlined residues failed to react with mAb 14. In C,protein extracts of HEK 293 cells transfected with individualpcDNA3-FLAG-GPBPΔ1 (Δ1)-pCDNA3-FLAG-GPBPΔ4 (Δ4), were analyzed bySDS-PAGE and Western blot with the indicated antibodies. Similar resultswere obtained for remaining antibodies included in the Table 1 underregion 7-8: N4, N7, N9, N11, N14, N25, N27, N28 (similar to N22); andN2, N3, N5, N10, N12, N13 (similar to N8). The N16 antibody was notmapped.

FIG. 19 shows the sequence of 91 kD GPBP (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used in this application, the term “native protein” means the proteinnaturally produced by the cell, including any post-translationalmodifications (PTMs), and includes non-denatured protein, or denaturedprotein (as, for example, naturally produced protein substantiallypurified and subjected to one or more denaturing agents to, for example,run on a SDS-PAGE gel).

As used in this application, “substantially purified polypeptide” meansthat the polypeptide has been separated from its in vivo cellularenvironments. It is further preferred that the isolated polypeptides arealso substantially free of gel agents, such as polyacrylamide, agarose,and chromatography reagents.

Unless clearly indicated otherwise by the context, embodiments disclosedfor one aspect of the invention can be used in other aspects of theinvention as well, and in combination with embodiments disclosed inother aspects of the invention.

In a first aspect, the present invention provides isolated polypeptidesof 90% or greater purity consisting of the amino acid sequence of SEQ IDNO: 2 (91 kD GPBP). The inventors have determined that the hypothesizedsequence of 91 kD GPBP previously proposed in WO 2004/070025 isincorrect, and have now isolated native 91 kD protein and determined itscorrect amino acid sequence, which is shown in SEQ ID NO:2. FIG. 19shows the sequence of 91 kD GPBP, and in bold cursive underlined form,and from N to the C terminus, the amino acid residues comprising theepitopes of Ab 24, mAb 14 and mAb e26 respectively. The first residue(Met) of canonical 77-kDa GPBP (SEQ ID NO:4) is highlighted in bold andboxed in the figure. Thus, 91-kDa and 77-kDa GPBP are identical in aminoacid sequence from the highlighted “Met” residue through the end of theprotein. As noted below, the inventors have obtained compelling evidencethat the mRNA of GPBP undergoes canonical (AUG) and noncanonical (ACG)translation initiation to generate two primary polypeptides of 77- and91-kDa, respectively. The results from this study also support that bothproducts enter the secretory pathway. However, whereas the 77-kDareaches the extracellular compartment and exists in a solubleimmunoprecipitable form, the 91-kDa remains mainly insoluble, associatedwith cellular membranes and likely reaches the external side of plasmamembrane. The evidence supports that the 120-kDa GPBP isoform is acovalently-derived product of the 91-kDa GPBP (ie: the only differencesare post-translational modifications) and thus shares the amino acidsequence of 91-kDa polypeptide. Therefore, as used herein, the term“91-kDa GPBP” includes the 91-kDa and post translational modificationsthereof, including but not limited to 120-kDa GPBP and aggregates of91-kDa and 120-kDa GPBP. The present invention provides additionalevidence for the 91-kDa GPBP to exist in a soluble form in the plasmaand urine revealing that the 91-kDa GPBP can be released from thecellular membranes. The polypeptides of this aspect of the invention canbe used, for example, to produce antibodies against 91-kDa GPBP, and astargets for identification of compounds that interfere with GPBPactivity, making them useful therapeutics for various disorders,including Goodpasture Syndrome.

Thus, our data support the notion that mRNA alternative translationinitiation is a strategy to direct GPBP to multiple locations includingsecretory pathway, plasma membrane and extracellular compartment.

In this aspect and the other polypeptide aspects and embodiments of theinvention, the polypeptides can be used, for example, to generatespecific antibodies for detection of different isoforms of native GPBPpresent in serum or in urine, which can thus be used as, for example,diagnostic agents for autoimmune and other disorders. The polypeptidesof the invention can also be used, for example, as tools to identifycandidate compounds for inhibiting various specific types of native GPBPisoforms and also to identify candidate compounds for treating, forexample, autoimmunity and protein misfolding-mediated disorders, asdiscussed in more detail below.

As used herein, “90% or greater purity” means that contaminatingproteins make up no more than 10% of the isolated polypeptide; invarious preferred embodiments, no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, or 0.5% of the isolated polypeptide (e.g., isolated polypeptidesof 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greaterpurity consisting of the amino acid sequence of SEQ ID NO: 2). It isfurther preferred that the isolated polypeptides are also substantiallyfree of gel agents, such as polyacrylamide and agarose. In a furtherpreferred embodiment, the isolated polypeptides are present in solution,frozen, or as a dried powder. In one preferred embodiment, the isolatedpolypeptides of this first aspect are optionally labeled with adetectable, non-polypeptide label, including but not limited tofluorescent labels or radioactive labels.

In a second aspect, the present invention provides substantiallypurified recombinant polypeptides comprising or consisting of thegeneral formula X-SEQ ID NO:2, wherein X is a detectable polypeptide. Inthis aspect, the correct amino acid sequence for 91 kD GPBP (SEQ IDNO:2) is expressed as a fusion protein with a detectable polypeptide.The polypeptides of this aspect of the invention can be used, forexample, to track 91 kD GPBP in cells, and as a detectable target foridentification of compounds that interfere with GPBP activity, makingthem useful therapeutics for various disorders, including GoodpastureSyndrome. As used in this aspect, a “recombinant polypeptide” means thatthe detectable polypeptide is not derived from GPBP or expressed from aGPBP mRNA, and thus fuses a heterologous detectable peptide with thecorrect 91 kD GPBP polypeptide. As used herein, a “detectablepolypeptide” is any heterologous peptide that can be detected, thuspermitting detection of the recombinant polypeptide. In one preferredembodiment, the detectable polypeptide comprises a fluorescent protein.Any fluorescent protein known in the art can be used in the invention.For example, green fluorescent proteins of cnidarians, which act astheir energy-transfer acceptors in bioluminescence, are suitablefluorescent proteins for use in the fluorescent indicators. A greenfluorescent protein (“GFP”) is a protein that emits green light, and ablue fluorescent protein (“BFP”) is a protein that emits blue light.GFPs have been isolated from the Pacific Northwest jellyfish, Aequoreavictoria, the sea pansy, Renilla reniformis, and Phialidium gregarium.See, Ward, W. W., et al., Photochem. Photobiol., 35:803 808 (1982); andLevine, L. D., et al., Comp. Biochem. Physiol., 72B:77 85 (1982). Avariety of Aequorea-related GFPs having useful excitation and emissionspectra have been engineered by modifying the amino acid sequence of anaturally occurring GFP from Aequorea victoria. See, Prasher, D. C., etal., Gene, 111:229 233 (1992); Heim, R., et al., Proc. Natl. Acad. Sci.,USA, 91:12501 04 (1994); U.S. Ser. No. 08/337,915, filed Nov. 10, 1994;International application PCT/US95/14692, filed Nov. 10, 1995; and U.S.Ser. No. 08/706,408, filed Aug. 30, 1996. The cDNA of GFP can beconcatenated with those encoding many other proteins; the resultingfusions generally are fluorescent and retain the biochemical features ofthe partner proteins. See, Cubitt, A. B., et al., Trends Biochem. Sci.20:448 455 (1995). Mutagenesis studies have produced GFP mutants withshifted wavelengths of excitation or emission. See, Heim, R. & Tsien, R.Y. Current Biol. 6:178 182 (1996). Suitable pairs, for example ablue-shifted GFP mutant P4-3 (Y66H/Y145F) and an improved green mutantS65T can respectively serve as a donor and an acceptor for fluorescenceresonance energy transfer (FRET). See, Tsien, R. Y., et al., Trends CellBiol. 3:242 245 (1993).

In another preferred embodiment of this second aspect, the detectablepolypeptide comprises a non-GPBP epitope for which antibodies arecommercially available, including but not limited to the FLAG (SigmaChemical, St. Louis, Mo.), myc (9E10) (Invitrogen, Carlsbad, Calif.),6-His (Invitrogen; Novagen, Madison, Wis.), glutathione S-transferase(GST) (Santa Cruz Biotechnology, Santa Cruz, Calif.), and HA(hemaglutunin) (Boehringer Manheim Biochemicals).

In all of the embodiments of the second aspect of the invention, theisolated polypeptide may preferably further comprise a linker sequencebetween the detectable polypeptide and the polypeptide of SEQ ID NO:2.In this embodiment, the linker is not a portion of GPBP or encoded by aGPBP mRNA. Such a linker can be of any desirable length, and preferablyis between 1 and 20 amino acids, if present; more preferably between 1and 15, 1-10, 1-5, 1-4, 1-3, or 1-2 amino acids, if present. The linkercan be used, for example, to optimally position the detectablepolypeptide and the 91 kD GPBP sequence and to include specific sequencefor protease recognition site to allow removal of detectablepolypeptide. In all of the embodiments of the second aspect of theinvention, the isolated polypeptide may further comprise any additionalresidues necessary for expression, such as an N-terminal methionineresidue or peptide sequences to deliver the polypeptide to differentcellular and extracellular compartments.

The substantially purified polypeptides of the invention can be made byany method known to those of skill in the art, but are preferably madeby recombinant means based on the teachings provided herein. Forexample, a coding region of interest as disclosed herein can be clonedinto a recombinant expression vector, which can then be used totransfect a host cell for recombinant protein production by the hostcells.

In a third aspect, the present invention provides substantially purifiednucleic acids encoding a polypeptide consisting of the amino acidsequence of SEQ ID NO:2 (91 kD GPBP). The substantially purified nucleicacid sequence may comprise RNA or DNA. As used herein, “substantiallypurified nucleic acids” are those that have been removed from theirnormal surrounding nucleic acid sequences in the genome or in cDNAsequences. Such substantially purified nucleic acid sequences maycomprise additional sequences useful for promoting expression and/orpurification of the encoded protein, including but not limited to polyAsequences, modified Kozak sequences, and sequences encoding epitopetags, export signals, and secretory signals, nuclear localizationsignals, and plasma membrane localization signals. In one preferredembodiment, the substantially purified nucleic acid coding regionconsists of the nucleic acid of SEQ ID NO:1, or a mRNA product thereof.In another preferred embodiment, the present invention providessubstantially purified nucleic acids encoding the polypeptide of anyembodiment of the substantially purified recombinant polypeptidescomprising or consisting of the general formula X-SEQ ID NO:2, asdiscussed in the second aspect of the invention.

In a fourth aspect, the present invention provides recombinantexpression vectors comprising the substantially purified nucleic acid ofany aspect of the invention operatively linked to a promoter.“Recombinant expression vector” includes vectors that operatively link anucleic acid coding region or gene to any promoter capable of effectingexpression of the gene product. The promoter sequence used to driveexpression of the disclosed nucleic acid sequences in a mammalian systemmay be constitutive (driven by any of a variety of promoters, includingbut not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven byany of a number of inducible promoters including, but not limited to,tetracycline, ecdysone, steroid-responsive). The construction ofexpression vectors for use in transfecting prokaryotic cells is alsowell known in the art, and thus can be accomplished via standardtechniques. (See, for example, Sambrook, Fritsch, and Maniatis, in:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed.E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.). The expression vector must be replicablein the host organisms either as an episome or by integration into hostchromosomal DNA. In a preferred embodiment, the expression vectorcomprises a plasmid. However, the invention is intended to include otherexpression vectors that serve equivalent functions, such as viralvectors.

In a fifth aspect, the present invention provides host cells that havebeen transfected with the recombinant expression vectors disclosedherein, wherein the host cells can be either prokaryotic or eukaryotic.The cells can be transiently or stably transfected. Such transfection ofexpression vectors into prokaryotic and eukaryotic cells can beaccomplished via any technique known in the art, including but notlimited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc.New York, N.Y.).

In a sixth aspect, the present invention provides a substantiallypurified polypeptide comprising the amino acid sequence of SEQ ID NO:2(91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP), wherein the polypeptide of SEQID NO:2 or SEQ ID NO:4 comprises one or more post-translationalmodifications (PTMs) directly and/or indirectly involving amino acidsresidues 305-344 GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO:10) (numbering based on position within 77 kD GPBP). As disclosed in theexamples that follow, the inventors provide the first purification ofnative 77 and 91 kD GPBP and have determined that existing monoclonalantibodies that bind to recombinant versions of 77 kD- and 91 kD-GPBP donot bind to purified native versions, verifying that structuraldifferences exist between recombinant and native forms of the 77 kD GPBPand between recombinant and native forms of the 91 kD GPBP. Thepolypeptides of this aspect of the invention can be used, for example,to produce antibodies against native GPBP forms, and as targets foridentification of compounds that interfere with native GPBP activity,making them useful therapeutics for various disorders, includingGoodpasture Syndrome. In one preferred embodiment, the one or more PTMscomprise covalent PTMs. In another preferred embodiment, the one or morePTMs comprise covalent PTMs within amino acids 305-344 (SEQ ID NO: 10).In one preferred embodiment the one or more PTMs directly or indirectlyinvolve residues 320-327 (EEFFDAVE, SEQ ID NO:5). In another preferredembodiment, the one or more PTMs comprise covalent PTMs within residues320-327 (EEFFDAVE, SEQ ID NO:5) (numbering based on position within 77kD GPBP). In another preferred embodiment, the one or more PTMs compriseone more PTMs present in residue 320, 321, and/or 327; most preferably,the one or more PTMs present at these residues comprise covalent PTMs.In a further preferred embodiment of any of the embodiments of thisaspect, the substantially purified polypeptide possesses an amino acidsequence consisting of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kDGPBP).

As used herein, the term “post-translational modification” (PTM) means amodification in the structure of a protein after its translation. In onepreferred embodiment, the PTM comprises addition of a functional group,including but not limited to carboxylation, methylation, citrullination,phosphorylation, glycosylation, and formation of atypical isoaspartyl.In another preferred embodiment, the PTM comprises an isomerization,leading to a conformational change.

As used herein, “directly” means that the PTM occurs within thespecified residues, while “indirectly” means that the PTM occurs outsidethe specified residues, but results in a structural change within thecited residues.

Any suitable method for making the covalently modified polypeptide ofSEQ ID NO:2 or SEQ ID NO:4 based on the teachings of the presentdisclosure can be used, including isolating from natural sources of GPBPas disclosed herein, and recombinant production of GPBP followed bysuitable covalent modification within the relevant region of amino acidresidues, using standard methods known to those of skill in the art.

In a seventh aspect, the present invention provides substantiallypurified polypeptides comprising the amino acid sequence of SEQ ID NO:2(91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP), wherein the polypeptide of SEQID NO:2 or SEQ ID NO:4 comprises one or more PTMs directly and/orindirectly involving residues 371-396 PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ IDNO:9) (numbering based on positions within 77 kD GPBP). As disclosed inthe examples that follow, the inventors provide the first purificationof native 77 kD and 91 kD GPBP and have determined that existingmonoclonal antibodies that bind to recombinant version of 77 kD and 91kD GPBP do not bind to the purified native 77 kD and 91 kD GPBPversions, verifying that structural differences exist betweenrecombinant and native forms of the 77 and 91 kD GPBP. The polypeptidesof this aspect of the invention can be used, for example, to produceantibodies against native GPBP, and as targets for identification ofcompounds that interfere with native GPBP activity, making them usefultherapeutics for various disorders, including Goodpasture Syndrome. Inone preferred embodiment, the one or more PTMs comprise covalent PTMs.In another preferred embodiment, the one or more PTMs comprise covalentPTMs within amino acids 371-396 (SEQ ID NO:9). In one preferredembodiment, the one or more PTMs directly or indirectly involve residues388-392 (DDVHR, SEQ ID NO:6). In another preferred embodiment, the oneor more PTMs comprise one or more covalent PTMs within residues 388-392(SEQ ID NO:6) In another preferred embodiment, the polypeptide furthercomprises one or more PTMs directly or indirectly involving residues320-327 (EEFFDAVE, SEQ ID NO:5) In a further preferred embodiment, theone or more PTMs within residues 320-327 are covalent PTMs. In variouspreferred embodiments of this aspect, the substantially purifiedpolypeptide possesses an amino acid sequence consisting of SEQ ID NO:2(91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP). Any suitable method for makingthe covalently modified polypeptide of SEQ ID NO:2 or SEQ ID NO:4 can beused, including isolating from natural sources of GPBP as disclosedherein, and recombinant production of GPBP followed by suitable covalentmodification within the relevant region of amino acid residues, usingstandard methods known to those of skill in the art.

In an eighth aspect, the present invention provides substantiallypurified monoclonal antibodies that selectively bind to thesubstantially purified polypeptides of the sixth or seventh aspect ofthe invention. As disclosed above, the inventors have for the first timeisolated native 77- and 91 kD GPBP species that when substantiallypurified do not bind to existing GPBP-specific monoclonal antibodies.For example, existing monoclonal antibodies do not detect GPBP in plasmaor urine samples in ELISAs, nor are they capable of use for purificationof plasma or urine GPBP. Thus, the monoclonal antibodies of theinvention are useful, for example, in ELISA-based assays for GPBPdetection in urine or plasma, and for purification of GPBP from plasmaor serum. The inventors further demonstrate herein that these native 77kD GPBP and native 91 kD GPBP species are pos-translationally modified,and that at least some of these PTMs render substantially purified,native GPBP non-reactive to existing monoclonal GPBP antibodies.Exemplary monoclonal antibodies according to this aspect of theinvention are provided in the examples that follow.

The “monoclonal antibodies” of the invention can be any type ofmonoclonal antibody, including but not limited to standard monoclonalantibodies, humanized monoclonals, chimeric monoclonals, and fragmentsthereof.

As used herein, “substantially purified” means that the recitedmonoclonal antibodies make up at least 80% of the antibodies in asubstantially purified sample; more preferable at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

As used herein, “selectively bind” means preferential binding of theGPBP monoclonal antibody to native GPBP epitope, as opposed to one ormore other biological molecules, structures, cells, tissues, etc., as iswell understood by those of skill in the art.

Monoclonal antibodies can be produced by obtaining spleen cells from theanimal [See Kohler and Milstein, Nature 256, 495-497 (1975)]. In oneexample, monoclonal antibodies (mAb) of interest are prepared byimmunizing inbred mice with native 77 kD GPBP, native 91 kD GBPB, or anantigenic fragment thereof, including, but not limited to, one or moreepitopes comprising or consisting of the PTM-containing peptidesEEFFDAVE (SEQ ID NO:5), DDVHR (SEQ ID NO:6), LINEEEFFDAVEAALDRQ (SEQ IDNO:8), PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ ID NO:9), andGGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO: 10). Thus, in afurther preferred embodiment, the monoclonal antibodies bind one or moreepitopes comprising one or more PTMs, selected from the group consistingof PTM-containing EEFFDAVE (SEQ ID NO:5), DDVHR (SEQ ID NO:6),LINEEEFFDAVEAALDRQ (SEQ ID NO:8), PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ IDNO:9), and GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQSQSEK (SEQ ID NO: 10). Ina further preferred embodiment, the one or more PTMs are covalent PTMs.In another preferred embodiment, the monoclonal antibodies bind to anepitope that comprises one or more PTMs (preferably covalent PTMs)present in residue 320, 321, and/or 327 (numbering based on 77 kD GPBP).

In one exemplary embodiment, the mice are immunized by the IP or SCroute in an amount and at intervals sufficient to elicit an immuneresponse. The mice receive an initial immunization on day 0 and arerested for about 3 to about 30 weeks Immunized mice are given one ormore booster immunizations of by the intravenous (IV) route.Lymphocytes, from antibody positive mice are obtained by removingspleens from immunized mice by standard procedures known in the art.Hybridoma cells are produced by mixing the splenic lymphocytes with anappropriate fusion partner under conditions which will allow theformation of stable hybridomas. The antibody producing cells and fusionpartner cells are fused in polyethylene glycol at concentrations fromabout 30% to about 50%. Fused hybridoma cells are selected by growth inhypoxanthine, thymidine and aminopterin supplemented Dulbecco's ModifiedEagles Medium (DMEM) by procedures known in the art. Supernatant fluidsare collected from growth positive wells and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay.Hybridoma cells from antibody positive wells are cloned by a techniquesuch as the soft agar technique of MacPherson, Soft Agar Techniques, inTissue Culture Methods and Applications, Kruse and Paterson, Eds.,Academic Press, 1973.

“Humanized monoclonal antibodies” refers to monoclonal antibodiesderived from a non-human monoclonal antibody, such as a mouse monoclonalantibody. Alternatively, humanized monoclonal antibodies can be derivedfrom chimeric antibodies that retains, or substantially retains, theantigen-binding properties of the parental, non-human, monoclonalantibodies but which exhibits diminished immunogenicity as compared tothe parental monoclonal antibody when administered to humans. Forexample, chimeric monoclonal antibodies can comprise human and murineantibody fragments, generally human constant and mouse variable regions.Humanized monoclonal antibodies can be prepared using a variety ofmethods known in the art, including but not limited to (1) graftingcomplementarity determining regions from a non-human monoclonal antibodyonto a human framework and constant region (“humanizing”), and (2)transplanting the non-human monoclonal antibody variable domains, but“cloaking” them with a human-like surface by replacement of surfaceresidues (“veneering”). These methods are disclosed, for example, in,e.g., Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc.Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv.Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536(1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol.31(3):169-217 (1994); and Kettleborough, C. A. et al., Protein Eng.4(7):773-83 (1991).

Monoclonal antibodies can be fragmented using conventional techniques,and the fragments screened for utility in the same manner as for wholeantibodies. For example, F(ab′)₂ fragments can be generated by treatingantibody with pepsin. The resulting F(ab′)₂ fragment can be treated toreduce disulfide bridges to produce Fab′ fragments. Fab fragments can beobtained by treating an IgG antibody with papain; F(ab′) fragments canbe obtained with pepsin digestion of IgG antibody. A F(ab′) fragmentalso can be produced by binding Fab′ described below via a thioetherbond or a disulfide bond. A Fab′ fragment is an antibody fragmentobtained by cutting a disulfide bond of the hinge region of the F(ab′)2.A Fab′ fragment can be obtained by treating a F(ab′)2 fragment with areducing agent, such as dithiothreitol. Antibody fragment peptides canalso be generated by expression of nucleic acids encoding such peptidesin recombinant cells (see, e.g., Evans et al., J. Immunol. Meth. 184:123-38 (1995)). For example, a chimeric gene encoding a portion of aF(ab′)2 fragment can include DNA sequences encoding the CH1 domain andhinge region of the H chain, followed by a translational stop codon toyield such a truncated antibody fragment molecule.

Examples of monoclonal antibody fragments include (i) a Fab fragment, amonovalent fragment consisting essentially of the VL, VH, CL and CH Idomains; (ii) F(ab)₂ and F(ab′)2 fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting essentially of the VH and CH1domains; (iv) a Fv fragment consisting essentially of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists essentially of a VH domain;and (vi) one or more isolated CDRs or a functional paratope.

To generate an antibody response, the immunogens are typicallyformulated with a pharmaceutically acceptable carrier for parenteraladministration. Such acceptable adjuvants include, but are not limitedto, Freund's complete, Freund's incomplete, alum-precipitate, water inoil emulsion containing Corynebacterium parvum and tRNA. The formulationof such compositions, including the concentration of the polypeptide andthe selection of the vehicle and other components, is within the skillof the art.

In a ninth aspect, the present invention provides substantially purifiedmonoclonal antibodies that specifically binds to the polypeptide of SEQID NO:2 and not to the polypeptide of SEQ ID NO:4. Such monoclonalantibodies of the invention are useful, for example, in distinguishing91 kD GPBP from 77 kD GPBP in assays including, but not limited to,ELISA-based assays for GPBP detection in urine or plasma. Suchmonoclonal antibodies can be generated using methods disclosed above andthe use of peptide immunogens present in the polypeptide of SEQ ID NO:2but not present in SEQ ID NO:4. Such immunogens may be of any suitablelength to generate an antibody response. In one exemplary embodiment,the monoclonal antibodies are generate against an immunogen comprisingor consisting of DGWKGRLPSPLVLLPRSARC (SEQ ID NO:7). Thus, in thisembodiment, the monoclonal antibody binds to an epitope within the aminoacid sequence DGWKGRLPSPLVLLPRSARC (SEQ ID NO:7). An exemplary suchantibody, Ab24, is disclosed below.

In a further aspect, the present invention provides isolated hybridomacells expressing the monoclonal antibodies of the eighth or ninthaspects of the invention.

The invention also provides methods for making the antibodies of theinvention, as disclosed above and below.

In a tenth aspect, the present invention provides methods for detectingcirculating Goodpasture antigen-binding protein (GPBP), comprising (a)contacting a plasma sample with a GPBP-binding molecule that binds toGPBP under conditions to promote selective binding of the GPBP-bindingmolecule to the GPBP;

(b) removing unbound GPBP-binding molecules; and

(c) detecting complex formation between the GPBP-binding molecule andthe GPBP in the plasma sample.

A “plasma sample” means blood plasma, the liquid component of blood, andis prepared, for example, by centrifugation of whole blood to removeblood cells. As used herein, a plasma sample also includes a blood serumsample, in which blood clotting factors have been removed.

In an eleventh aspect, the present invention provides methods fordetecting urinary Goodpasture antigen-binding protein (GPBP), comprising(a) contacting a urine sample with a GPBP-binding molecule that binds toGPBP under conditions to promote selective binding of the GPBP-bindingmolecule to GPBP;

(b) removing unbound GPBP-binding molecule; and

(c) detecting complex formation between the GPBP-binding molecule andthe GPBP in the urine sample.

Urine samples are easily obtained, and analyte determination in urine iswell known in the art.

A “GPBP-binding molecule” is a peptide or nucleic acid molecule thatbinds selectively to GPBP, as opposed to one or more other biologicalmolecules, structures, cells, tissues, etc. Exemplary embodiments ofsuch GPBP-binding molecules include but are not limited to antibodies,aptamers or substrates. As used herein, a “GPBP substrate” is a targetof GPBP biological activity that binds to GPBP, or a fragment thereofthat retains GPBP-binding activity. Such GPBP substrates include, butare not limited to, 1-20 (SEQ ID NO:16), GPBP-interacting proteins(GIPs) (SEQ ID NOS:17-21), myelin basic protein (MBP) and derivativesthereof (SEQ ID NOS:22-25), prion protein (PrP) (SEQ ID NO:26), type IVcollagen α3 chain NC1 domain (α3(IV)NC1) (SEQ ID NO:27), and Alzheimer'sdisease beta peptide (Aβ₁₋₄₂) (SEQ ID NO:28). Exemplary referencesdemonstrating GPBP binding of these substrates can be found in U.S. Pat.Nos. 6,579,969; 7,147,855; and 7,326,768, incorporated by referenceherein in their entirety.

As disclosed in the examples that follow, the inventors have discoveredcirculating and urinary forms of GPBP, including GPBP isoforms of 160-,91-, 77-, 70-, 66-, 60-, 58-, 56- 53- 50- 46- 35 and 34-kD, and variousaggregates thereof. Thus, in the tenth and eleventh aspects, the term“GPBP” refers to all GPBP isoforms reactive with GPBP-selectiveantibodies, including but not limited to 77 kD GPBP and 91 kD GPBP, aswell lower and higher molecular weight GPBP isoforms of 160-, 60-, 58-,56- 53- 50- 46- 35 and 34-kD, and aggregates thereof.

The “plasma sample” or “urine sample” may be obtained from any suitablesubject, preferably from a mammal, including but not limited to a human,dog, cat, horse, or livestock (cow, sheep, etc.). In a most preferredembodiment, the plasma sample or urine sample is obtained from a humansubject, such as a human subject suspected of having an autoimmunecondition including but not limited to Goodpasture Syndrome and/orimmune-complex mediated glomerulonephritis. As disclosed herein, theinventors have isolated native circulating 77 kD GPBP from human plasmaand have observed increased levels in Goodpasture patients and in animalmodels for immune complex-mediated glomerulonephritis, demonstratingthat GPBP secretion occurs in vivo and revealing the clinical utility ofserological and urinary determination of GPBP.

The antibody can be any selective GPBP antibody, whether polyclonal,monoclonal, or humanized monoclonal as described above, althoughmonoclonal antibodies are preferred. In one embodiment, antibodiesaccording to the eighth or ninth aspects of the invention are used. Themethods of the tenth and eleventh aspect of the invention may compriseanalyzing a specific GPBP isoform, such as 77 kD GPBP or 91 kD GPBP; inthese embodiments, antibodies selective for 77 kD GPBP or selective for91 kD GPBP can be used, including but not limited to those selectiveantibodies disclosed herein. In a most preferred embodiment, theantibodies for use in the methods of the tenth and eleventh aspects ofthe invention are those that bind to native GPBP isoforms, such as thosedisclosed herein.

Conditions suitable to promote binding of GPBP-binding molecules, suchas antibodies, aptamers or substrates, to GPBP in the plasma or urinesamples can be determined by those of skill in the art based on theteachings herein and the examples provided below. For example,antibody-antigen binding often depends on hydrophobic interactions (theso called hydrophobic bonds); thus, high salt concentrations, such as inthe molar range can be used to reduce nonspecific binding and increasespecific antigen-antibody binding. Optionally, further steps may beincluded to promote selectivity and specificity, including but notlimited to one or more wash steps to remove unbound or weakly boundserum proteins; inhibitors of non-specific binding to reduce binding ofhigh concentration serum proteins, control samples known to contain GPBPisoforms and/or negative controls known not to bind to GPBP isoforms,and/or inclusion of serum or urine samples known to not possess GPBP(ex: deleted for GPBP).

These tenth and eleventh aspects of the present invention may be used totest for the presence of GPBP in the plasma or urine sample by standardtechniques including, but not limited to ELISA, immunoflourescence, andchromatography (for example, lateral flow assays where the antibody isimmobilized on a surface and plasma or urinary proteins are labeled andallowed to flow over the surface under conditions suitable to permitbinding of the antibody to GPBP in the plasma or urine). In oneembodiment, functional beads (Becton Dickinson technology) coupled toflow cytometry are used; this technique is an emerging method to measurethe levels of proteins in biological fluid or cell/tissue extracts.Specifically, beads made of a fluorescence matrix are coated with one ormore specific GPBP antibodies, mixed with the plasma sample and furtherincubated with a detecting antibody labeled with a phycoerythrins.Finally, beads are analyzed by a flow cytometry program which selectsthe beads according matrix fluorescence emission and measurement of thelevel of the analyte through phycoerythrin emission. There are up tothirty different types of beads that can be simultaneously detected anddiscriminated by the cytometer. This method couples high sensitivity andperformance with versatility since a specific bead type coated with GPBPantibody can be mixed with a distinct bead type coated with bindingpeptides for other analyte (i.e. autoantibodies) and simultaneouslymeasured. The measurement of various analytes could enhance thepotential of GPBP determination. In one embodiment, the techniques maydetermine only the presence or absence of the GPBP isoform(s).Alternatively, the techniques may be quantitative, and provideinformation about the relative amount of the protein or peptide ofinterest in the sample. For quantitative purposes, ELISAs are preferred.

Detection of immunocomplex formation can be accomplished by standarddetection techniques. For example, detection of immunocomplexes can beaccomplished by using labeled antibodies or secondary antibodies. Suchmethods, including the choice of label are known to those ordinarilyskilled in the art. (Harlow and Lane, Supra). Alternatively, theantibodies can be coupled to a detectable substance. The term “coupled”is used to mean that the detectable substance is physically linked tothe antibody. Suitable detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase. Examples of suitable prosthetic-group complexesinclude streptavidin/biotin and avidin/biotin. Examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin. An example of a luminescent material includesluminol Examples of suitable radioactive material include ¹²⁵I, ¹³¹I,³⁵S or ³H.

As noted above, the inventors have observed increased levels inGoodpasture patients and in animal models for immune complex-mediatedglomerulonephritis, demonstrating that GPBP secretion occurs in vivo andrevealing the clinical utility of serological determination of GPBP.Thus, the methods of this aspect of the invention can be used, forexample, to detect GPBP-mediated disorder in a subject, including butnot limited to an antibody-mediated disorder (including but not limitedto a glomerulonephritis selected from the group consisting of IgAnephropathy, systemic lupus erythematosus and Goodpasture disease),inflammation, an ER-stress mediated disorder, and drug-resistant cancer.In these embodiments, the methods would comprise comparison of GPBPlevels detected in a test serum or urine sample with a control, such asa control from a serum or urine sample known to have “normal” levels ofGPBP or previously determined normal values for GPBP in sera or urinefrom the subject from whom the serum is obtained. In variousembodiments, the control provides a standard curve using recombinantGPBP or a reference value. In comparing the amount of GPBP in the serumor urine sample to a control, an increase (preferably a statisticallysignificant increase using standard statistical analysis techniques) inGPBP in the serum or urine sample relative to the control indicates thepresence of one or more of the disorders noted above, or an increasedrisk of developing one or more of the disorders, all of which arecorrelated with increased GPBP expression.

It has previously been disclosed that increased GPBP expression inducesIgA nephropathy, immune complex-related glomerulonephritis; thatincreased GPBP expression is intimately involved in Goodpasture Syndromepathogenesis; and that increased GPBP expression mediates resistance ofcancer cells to chemotherapeutic agents that induce protein misfoldingand ER stress-mediated cell death. The methods of the present inventionthus provide methods for diagnosing these disorders by serological orurine testing for the presence of GPBP. Thus, the methods identifyindividuals either having or at risk of being stricken with one or moreof an antibody-mediated disorder (including but not limited to aglomerulonephritis selected from the group consisting of IgAnephropathy, systemic lupus erythematosus and Goodpasture disease),inflammation, an ER-stress mediated disorder, and drug-resistant cancer.In one non-limiting embodiment, the methods can be used to test cancerpatients either prior to or after initiation of a chemotherapy regimen;those patients that test positive for increased serum levels of GPBP areat increased risk of having a drug-resistant tumor or of their tumor isdeveloping drug-resistance, and an attending physician can assessappropriate treatment options in light thereof. Furthermore, suchpatients may undergo periodic testing for serum or urine levels of GPBPto monitor potential risk of developing a drug-resistant tumor.Similarly, patients thought to be at risk for developing, or suspectedof already having developed a glomerulonephritis selected from the groupconsisting of IgA nephropathy, systemic lupus erythematosus andGoodpasture disease, can be tested for serum or urine levels of GPBP.Further embodiments will be clear to those of skill in the art based onthe teachings herein.

GPBP is a circulating molecule and GBM (glomerular basement membrane) aprincipal component of the glomerular filtration barrier; therefore,GPBP accumulation in the glomerulus could result from local productionbut also from the sequestration of circulating GPBP produced elsewhere,and could also be reflected in increased GPBP in the urine. The localoverproduction could account for primary antibody-mediatedglomerulonephritis whereas increased circulating levels may inducesecondary forms of this pathology and perhaps are responsible fordisease recurrence upon renal transplantation. Consequently, in anotherembodiment, quantification of the levels of circulating or urinary GPBPis useful in discriminating primary from secondary antibody-mediatedglomerulonephritis and for the clinical monitoring of renaltransplantation.

In a further embodiment, combining GPBP determination with analysis ofother analytes the methods permit one to perform differential diagnosisor prognosis in the above disorders. In one non-limiting example, wehave found that some IgA nephropathy patients produce anti-basementmembrane autoantibodies. These circulating autoantibodies recognize theNC1 domain of type IV collagen. Determination of the titer of theseantibodies could help to monitor disease progression or also todistinguish different IgA nephropathy patients or to perform prognosisin these patients. By measuring anti-ssDNA, anti-nucleosomeautoantibodies and GPBP levels one can diagnose systemic lupuserythematosus but also distinguish between primary IgA nephropathy andIgA nephropathy secondary to systemic lupus erythematosus. In variousfurther embodiments, any determination used to diagnosis of primarydiseases listed in Donadio and Grande (2002) N Engl J Med 347, 738-748associated with glomerular deposition of IgA, can be used in conjunctionwith the methods of the invention for plasma or urinary detection ofGPBP for differential diagnosis in secondary IgA nephropathy patients.

In another embodiment, a normal value of GPBP as a reference for anstandard curve is between ˜1 ng/ml-10 ng/ml in plasma and approximately0.2 ng/ml to 1.5 ng/ml in urine, while Goodpasture patients exceed thenormal by at least 2-fold; in other embodiments, by at least 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or more the normal values.

In a twelfth aspect, the present invention provides methods forisolating native GPBP isoforms, comprising:

(a) subjecting a plasma sample to ammonium sulfate precipitation;

(b) conducting ion-exchange chromatography (IEC) on the ammonium sulfateprecipitated serum sample;

(c) identifying IEC fractions containing native GPBP isoforms;

(d) subjecting IEC fractions containing native GPBP isoforms to gelfiltration chromatography (GFC); and

(e) identifying GFC fractions containing native GPBP isoforms.

In one preferred embodiment, these methods can be used, for example, tosubstantially purify native 77 kD GPBP from plasma, as disclosed in moredetail in the examples that follow.

In a thirteenth aspect, the present invention provides methods forisolating native

GPBP isoforms, comprising:

(a) subjecting a urine sample to salt precipitation;

(b) conducting double ion-exchange chromatography (IEC) on the saltprecipitated protein sample; and

(c) identifying IEC fractions containing native GPBP isoforms.

As used herein, “double ion-exchange chromatography” means carrying outtwo successive and distinct ion-exchange chromatography steps prior tostep (c). Exemplary embodiments of IEC techniques are well known in theart, and include those disclosed in the examples that follow.

In one preferred embodiment, these methods can be used, for example, tosubstantially purify native 91 kD GPBP from urine, as disclosed in moredetail in the examples that follow.

In a fourteenth aspect, the present invention provides methods forisolating native GPBP isoforms, comprising:

(a) passing a plasma sample or urine sample through an affinity columncomprising a GPBP-binding molecule that selectively bind to native GPBP;

(b) washing unbound protein from the plasma or urine sample from theaffinity column; and

(c) eluting native GPBP isoforms from the column.

In one preferred embodiment, these methods can be used, for example, tosubstantially purify native 77 kD GPBP and native 91 kD GPBP from plasmaand urine, as disclosed in more detail in the examples that follow. Inanother preferred embodiment, the GPBP-binding molecule comprise GPBPantibodies. In another preferred embodiment, the antibodies comprise thenovel monoclonal antibodies of the present invention. In anotherpreferred embodiment, the eluting step comprises use of a denaturingeluting buffer.

Details of the purification methods of the twelfth, thirteenth, andfourteenth aspects of the invention are provided in the examples below.

Example 1 Summary

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thrkinase for the type IV collagen of basement membrane. More recently, wehave shown that GPBP is an extracellular protein that when overexpressedinduces type IV collagen disorganization and deposit of immune complexesin glomerular basement membrane (Ref 4). Here we show that cellsexpressed at least two GPBP isoforms resulting from canonical (77-kDa)and noncanonical (91-kDa) mRNA translation initiation. The 77-kDapolypeptide interacted with type IV collagen and localized as a solubleform in the extracellular compartment. The 91- and derived 120-kDapolypeptides associated with cellular membranes and regulated the levelsof the 77-kDa polypeptide in the extracellular compartment. The FFATmotif and the 26-residue Ser-rich region were required for theexportation of the 77-kDa polypeptide. And removal of the 26-residueSer-rich region yielded the previously recognized GPBP isoform(GPBPΔ26/CERT) that was cytosolic and in contrast to GPBP, sensitive tosphingomyelinase cell treatment. These and previous data implicateCOL4A3BP in a multi-compartmental program for protein secretion (i.e.type IV collagen) which includes: 1) phosphorylation and regulation ofprotein molecular/supramolecular organization (GPBP); and 2)inter-organelle ceramide trafficking and regulation of protein cargotransport to the plasma membrane (GPBPΔ26/CERT). Finally, we haveisolated circulating 77-kDa GPBP from human plasma and have observedincreased levels in Goodpasture patients and in animal models for immunecomplex-mediated glomerulonephritis, demonstrating that GPBP secretionoccurs in vivo and revealing the clinical utility of serologicaldetermination of GPBP.

Introduction

Goodpasture antigen-binding protein (GPBP) phosphorylates thenoncollagenous-1 (NC1) domain of the α3 chain of type IV collagen[α3(IV)NC1] (1). This domain is a pivotal structure in the molecular andsupramolecular organization of the glomerular basement membrane (GBM)collagen and also the target of autoantibodies mediatingglomerulonephritis in Goodpasture disease (2). Increased GPBP expressionhas been associated with autoimmune pathogenesis including Goodpasturedisease (3) and with the induction of GBM collagen disorganization anddeposit of IgA antibodies (4). These observations suggest that GPBPregulates GBM collagen organization and induces type IV collagen-basedantibody-mediated glomerulonephritis when its expression is abnormallyelevated (3, 4). COL4A3BP also encodes for GPBPΔ26, a more-abundantless-active alternatively spliced GPBP variant lacking a 26-residueSer-rich region, which is apparently not regulated under thesepathological conditions (3).

GPBP contains multiple structural elements including N terminalpleckstrin homology (PH) domain, Ser-Xaa-Yaa region, bipartite nuclearlocalization signal, coiled-coil domain, two phenylalalines in an acidictrack (FFAT) motif and C terminal steroidogenic acute regulatory relatedlipid transfer (START) domain. Additional structural features includemotifs for self-interaction and phosphorylation (1, 3, 5, 6). The PHdomains comprise a variety of poorly conserved structures present onlyin eukaryotes which have been proposed to mediate protein targeting tocellular membranes through interaction with phosphoinositides (7). Avariety of proteins including several protein kinases contain PH domains(8). The FFAT motifs target proteins to the ER through interaction withthe transmembrane cytosolic domain of the vesicle associated membraneprotein-associated proteins (VAPs) (9), which have been proposed to playa role in maintaining homeostasis for protein folding in the endoplasmicreticulum (ER) and in regulating protein cargo transport to the plasmamembrane (10, 11). The START domains bind lipids including ceramide,phospholipids and sterols, and are modules present in a variety ofproteins with distinct physiological and pathological functions (12,13).

Recent reports have implicated the FFAT motif and PH domain in thebinding of GPBP polypeptides to the ER and Golgi apparatus,respectively. The binding to these organelles has been postulated toenable the START domain to capture ceramide from the ER and to deliverit to the Golgi apparatus. Based on these observations, GPBPpolypeptides have been described as non-vesicular cytosolic ceramidetransporters and renamed CERT_(L) (GPBP) and CERT (GPBPΔ26) (5, 14).However, the conclusions of these authors were made in the absence ofprecise data related to the intracellular distribution of the nativeproteins and in complete disregard of immunochemical evidencedemonstrating predominant expression of GPBP in association withbasement membranes (3). More recent reports have shown thatCERT-dependent ceramide transport is critical for recruitment ofphospholipase A2α as well as for the recruitment and activation ofprotein kinase D at the trans Golgi network, thereby ultimatelyregulating prostaglandin production and protein exocytosis, respectively(6, 15).

Immunohistochemical evidence suggests that GPBP is primarilyextracellular, although with the potential to localize to variousintracellular sites (3, 4). Protein distribution is highly informativewith respect to protein function; therefore, additional studies wereneeded to understand the biological function of GPBP. Here wedemonstrate that the translation of the mRNA for GPBP generated severalpolypeptides, none of which were significantly expressed in the cytosol.On the contrary, the current study provides evidence that GPBP entersinto the secretory pathway and interacts with type IV collagen.Furthermore, we show that removal of 26-residue Ser-rich region byalternative exon splicing localizes the protein to the cytosol,revealing that GPBPΔ26/CERT represents a soluble, intracellular versionof GPBP. The present data suggest that alternative exon splicing andtranslation initiation are strategies to direct the products of COL4A3BPto different locations where they are expected to coordinate amulti-compartmental biological program. Various lines of evidencesupport that the later includes phosphorylation and regulation ofbasement membrane collagen organization (GPBP) (1, 3, 4) andinter-organelle ceramide transport which regulates vesicular proteincargo transport to the plasma membrane (GPBPΔ26/CERT) (6, 14). Finally,we show that 77-kDa GPBP is a serological component that may be used asa clinical marker of antibody-mediated glomerulonephritis (i.e.Goodpasture disease and immune complex-mediated glomerulonephritis).

Materials and Methods

Processing of serum samples—Mice and human blood samples were obtainedaccording to institutional guidelines for human studies and animalexperimentation. We used sera from New Zealand white (NZW) mice thatwere previously characterized (4) and which represent healthy young(4-month) and old undergoing IgA immune complex-mediated (7-month).Human plasmapheresis and sera from control or Goodpasture patients wereobtained following standard procedures.

Antibodies and recombinant proteins—Using truncated recombinant GPBPisoforms and synthetic peptides, we have mapped the epitope ofGPBP/GPBPΔ26-specific mouse monoclonal antibody 14 (mAb 14) (1) to theFFAT motif (FIG. 9). Mouse mAb e26 was raised against the 26-residuescharacteristic of GPBP (GPBPpep1) and therefore, was not reactive withGPBPΔ26/CERT (FIG. 1A). Human monoclonal F(ab)₂ fragments were isolatedfrom a recombinant F(ab)₂ expression library using a synthetic peptiderepresenting the alternatively translated region (ATR) of GPBP (FIG. 2C)(Antibodies by Design, MorphoSys AG). Reactive F(ab)₂ fragments werefurther characterized using Western blot and recombinant proteinsexpressing the predicted ATR (not shown). The most reactive F(ab)₂fragment (Ab 24) was used to characterize native GPBP polypeptides andthe least reactive F(ab)₂ fragment (Ab 20) was used as negative controlin these studies. The previously reported (4) immunopurified chickenpolyclonal GPBP-specific antibodies (αGPBP) were biotinylated for use inflow cytometry or labeled with Alexa Fluor 647 (Invitrogen) for directimmunofluorescence. Polyclonal antibodies specific for GPBP andGPBPΔ26/CERT were produced either in rabbits immunized withGST-FLAG-GPBP (1) following standard procedures (αGPBPr) or in chickensimmunized with a specific synthetic peptide and purchased from Abcam(αGPBPab). Specific antibodies in αGPBPr were affinity-purified usingrecombinant FLAG-GPBP (see below) bound to Sepharose-CNBr (Sigma). Forglyceraldehyde-3-phosphate dehydrogenase detection, we used a mousemonoclonal antibody provided by Erwin Knecht. Polyclonal antibodiesspecific for calregulin, p65 or cathepsin D were from Santa CruzBiotechnology Inc and those specific for pyruvate dehydrogenase (PDH)were from Molecular Probes. Monoclonal antibodies specific for PrP(clone 3F4) or for golgin-97 were from Clontech and Molecular Probes,respectively. To detect FLAG, we used FLAG/M2 or FLAG/M2-horseradishperoxidase (HRP) (Sigma) for Western blot analysis and chickenantibodies (αFLAG) or goat antibodies (αFLAG-FITC) forimmunofluorescences (Abeam). Alexa Fluor® 488-streptavidin was fromMolecular Probes and secondary antibodies were from Promega (anti-mouseand anti-rabbit HRP conjugates), Jackson Immunoresearch (anti-humanF(ab)₂—HRP) and Sigma (anti-chicken HRP and other FITC and TRITCconjugates). Recombinant FLAG-GPBP and FLAG-GPBPΔ26 were expressed inPichia pastoris and affinity-purified as previously described (1, 3).

Plasmid constructs—The production of pc-n4′, a pcDNA3(Invitrogen)-derived construct expressing a cDNA which contained the 5′untranslated region (UTR) and coding sequence of GPBP mRNA has beenreported (1). Plasmids derived from pc-n4′ included pc-GPBP-Met, adeletion mutant devoid of 5′UTR, and pc-n4′-Mmut, a construct where thecanonical AUG (Met) translation initiation was substituted with GGA(Gly). The production of pc-FLAG-GPBP, which expresses the FLAG sequencefused to the coding region of GPBP, was previously reported (1) and usedto obtain pc-FLAG-GPBP_(ΔFFAT), bearing a deletion in the FFAT motif(FIG. 9). The pc-FLAG-GPBPΔ26 expresses the FLAG sequence fused to thecoding region of GPBPΔ26 and has been produced similarly topc-FLAG-GPBP. To determine the initiation site that accounted for theATR, we produced pc-n4′ and pc-n4′-Mmut mutants by introducing stopcodons at various positions in the open reading frame (ORF) upstream ofiMet position. The pSilencer™ 2.1-U6 hygro (Ambion) was employed fortransient expression of small interfering mRNAs (siRNAs) specific forGPBP or for GPBP/GPBPΔ26. The corresponding derived constructs and cDNAtarget sequences were: pSi-GPBP/GPBPΔ26-2, ACAGAGTATGGCTGCAGAG (SEQ IDNO: 11); pSi-GPBP/GPBPΔ26-3, GTACTTTGATGCCTGTGCT (SEQ ID NO: 12);pSi-GPBP-1, GCCCTATAGTCGCTCTTCC (SEQ ID NO: 13). Selection of the targetsequence and plasmid construction were based on manufacturer'srecommendations. The efficiency of siRNA expressing-plasmids wasassessed in a cell recombinant expression system (not shown). Thecontrol plasmid in these studies (pSi-Control) was designed fortargeting the mRNA of green fluorescence protein, a protein notexpressed in human cells. All mutants were produced by standardPCR-based mutagenesis and the fidelity of all the cDNAs cloned wasconfirmed by nucleotide sequencing.

Cell culture and transfection—HEK-293 or HeLa cells were grown withDulbecco's modified Eagle's medium or Minimal Essential Medium Eaglerespectively, supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovineserum and penicillin (100 U/ml)/streptomycin sulfate (0.1 mg/ml), at 37°C. in a humidified 5% CO₂ environment. Unless otherwise indicated thecells used in the studies were HEK 293 cells.

Transfections were performed for 16-24 h using ProFection MammalianTransfection System-Calcium Phosphate (Promega) or Lipofectamine 2000(Invitrogen), following manufacturer's recommendations. Forimmunofluorescence studies, cells were seeded on poly-L-lysine-coatedcover slips in 24-well plates. When indicated HEK 293 cells weretransfected with pc-n4′-Mmut and selected with G418 (Invitrogen) for 15days. Resistant cells were further cloned by limiting dilution and theexpression of 91-kDa GPBP in a number of individual clones wasdetermined by Western blot analysis of cell extracts (see below). Clonesexpressing elevated (c8, c14) or reduced (c19) levels of 91-kDa wereused in functional studies.

In vitro transcription and translation—We used TNT® T7 CoupledReticulocyte Lysate System (Promega) to perform in vitrotranscription/translation of ˜1 μg of plasmid, following themanufacturer's recommendations. For assessing protein synthesis,[S³⁵]methionine was added to the mixtures and labeled polypeptides wereidentified by SDS-PAGE and fluorography. Briefly, after electrophoresisgels were fixed 1 h with 45% methanol and 7.5% acetic acid.Subsequently, gels were treated twice with dimethylsulfoxide for 30 minand with 22.5% of 2,5-dipheniloxazol in dimethylsulfoxide for additional30 min. Finally, gels were equilibrated with water, dried and exposed at−70° C.

Cell extracts and cell fractioning—To obtain cell extracts, growingcultures were rinsed with ice-cold phosphate buffered-saline (PBS) andhomogenized on ice bed with 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5%Triton X-100, 1 mM phenylmethylsulphonyl fluoride (PMSF) and 10 μg/mlleupeptin. Mixtures were cleared by centrifugation at 500×g for 10 min,protein concentration determined and stored at −70° C.

For subcellular fractionation, cultures at 90% confluence were collectedin PBS and subjected to centrifugation (500×g for 10 min). Cellularpellets were dispersed in 250 mM sucrose, 10 mM PBS pH 7.5 containing 10μg/ml leupeptin, 1 mM PMSF and disrupted with Dounce homogenization (20strokes) using a glass pestle. Cell homogenates were clearedprogressively by sequential centrifugation to obtain the different cellfractions. Nuclei and unbroken cells were collected by centrifugation at500×g for 10 min. The supernatant was further cleared by centrifugationat 7,000×g for 10 min to obtain mitochondrial/lysosome fraction.Finally, the supernatant was cleared by centrifugation at 150,000×g for1 h to obtain microsomal fraction which contains fragments of cellularmembranes i.e. endoplasmic reticulum, plasma membrane and secretoryvesicles (pellet) and the cytosolic fraction (supernatant). All stepswere performed at 0-4° C. and protein concentrations determined usingProtein Assay reagent (Bio-Rad).

For some purposes, the supernatant of 500×g was loaded on a resource-QFPLC column, and the bound material eluted in 0 to 1 M NaCl gradient in10 mM Tris-HCl pH 8.0. The 0.55-0.6 M NaCl fractions containing the bulkof cellular GPBP were precipitated with ethanol and used as partiallypurified GPBP for Western blot analysis.

Ex vivo cross-linking, sphingomyelinase treatment andFLAG-immunoprecipitation—For ex vivo cross-linking, we used HEK293-FLAG-α3(IV) cells expressing an exportable human α3(IV)NC1 domain(BM40-FLAG-α3(IV)NC1) which was obtained essentially as previouslyreported (1, 16). Cells were grown up to 70-90% of confluence in either150-mm plates (native GPBP) or six-well plates (recombinant GPBP).Cross-linking was performed 48 h after transfection or when cellsreached the indicated confluence. Briefly, cells were brought to RT byrinsing with PBS and incubated for 10 min with culture medium containing1% formaldehyde. The cross-link reaction was quenched with 125 mMGly-HCl in PBS (pH 7.4) for 10 min at RT. Cells were brought to 4° C. byrinsing with ice-chilled PBS and procedure continued at 4° C. Cells werelysed with 1 or 5 ml (six-well or 150-mm plate) of extraction buffer [16mM Tris-HCl pH 7.5, 160 mM NaCl, 2 mM ethylenediaminetetraacetic acid(EDTA), 1.1% Triton X-100, 0.01% SDS, 10 μg/ml leupeptin, 1 mM PMSF] for30 min, centrifuged at 500×g for 10 min to remove cell debris and thesupernatants were overnight extracted with 50 or 250 μl (six-well or150-mm plate) of a 50% slurry of αFLAG-affinity gel using gentlerocking. The beads were collected by centrifugation and washed twicewith 1 ml of extraction buffer and once with Tris-buffered saline (TBS,50 mM Tris-HCl pH 7.5, 150 mM NaCl). Proteins were eluted twice with 25or 125 μl (six-well or 150-mm plate) of a 100 μg/ml solution of FLAGpeptide in TBS at RT. Eluted samples were boiled with electrophoresissample buffer (2×) for 15 min to reverse cross-linking and furtheranalyzed by SDS-PAGE and either Coomassie blue staining or Western blot.

When indicated, HeLa cells transfected with pc-FLAG-GPBP orpc-FLAG-GPBPΔ26 were treated or not with Bacillus cereussphingomyelinase (Sigma) as previously described (5) and cells wereeither fixed with methanol/acetone and analysed by directimmunofluorescence (see below) or lysed in 10 mM Tris-HCl pH 7.5, 150 mMNaCl, 0.5% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM sodiumorthovanadate, 10 μg/ml leupeptin, 1 mM PMSF, cleared by centrifugation(500×g for 10 min) and used for FLAG-immunoprecipitation (see above).The immunopurified materials from untreated cells were divided andone-half was treated with 5 U/μl of λPPase (New England Biolabs) at 30°C. for 30 min following manufacturer's recommendations. All the sampleswere further analysed by Western blot using anti-FLAG antibodies.

For some experiments, cells were grown in 150-mm plates, transfectedwith 20 μg of plasmid constructs encoding FLAG-tagged proteins andcultured for two additional days in fresh media. Twenty milliliters ofmedia were used for FLAG-immunoprecipitation essentially as aboveindicated.

Flow cytometry—Cells were gently detached and dispersed in culturemedia. Non-specific antibody binding sites on cell surface were blockedwith mouse ascites fluid containing non-relevant mAb (blockingsolution). Cells were subsequently incubated in blocking solution in thepresence or absence of biotinylated αGPBP with or without blockingpeptide (GPBPpep1) or a non-relevant synthetic peptide. Cells wereincubated with Alexa Fluor® 488-streptavidin in blocking solution andfurther subjected to analysis in a Cytomics FC500 flow cytometer(Beckman Coulter) to measure fluorescence emission. Cell integrity wasassessed measuring forward and side scattering, using untreated freshcells as reference. All incubations were at RT for 1 h.

Direct and indirect immunofluorescence with fixed cell—Cells weretransfected and fixed with methanol-acetone (1/1) chilled at −20° C. for10 min. Subsequently, cells were incubated with blocking solution(rabbit serum diluted 1:2 in PBS) for 30 min at RT, incubated with theprimary antibodies (20 μg/ml in blocking solution) for 2 h at 37° C. ina humidified chamber, followed by incubation with the secondary antibody(1:200 in blocking solution) for 1 h at RT. Cells were stained with DAPI(1.25 μg/ml) in mounting fluid (DAKO) and visualized in an Axioskop-2plus microscope (Carl Zeiss) combined with a Spot camera and softwarev2.2 (Diagnostic Instruments). For some experiments, cells weretransfected, fixed, incubated with αFLAG-FITC and visualized as aboveindicated. Non-transfected cells were used as negative controls.

Direct immunofluorescence of living cells—Cells were cultured onglass-bottom microwell dishes (MatTek Corp) and when they reached ˜50%confluence, the media were discarded and replaced by fresh mediacontaining 10 μg/ml αGPBP-Alexa Fluor 647 with an excess of GPBPpep1 orequimolecular amounts of an unrelated synthetic peptide along withRhodamine 123 (Invitrogen) for mitochondrial staining of living cells.Live cell analysis of fluorescence was performed with a Leica TCS SP2inverted confocal microscope. Cells were maintained at 37° C. in ahumidified 5% CO₂ environment in all the steps.

Mass spectrometry—Individual protein bands were excised from Coomassieblue-stained gel, distained, in-gel trypsin digested, and centrifuged.One microliter of the supernatant was dried and resuspended with 1 μl ofmatrix solution (a-Cyano-4-hydroxycinnamic acid, from Sigma), applied tothe sample plate, dried and introduced into the mass spectrometer.Tryptic digests peptides were analyzed by MALDI/TOF/TOF massspectrometry (4700 Proteomics Analyzer, Applied Biosystems). Collecteddata were analyzed with GPS software (Applied Biosystems) and proteinidentification was carried out using the search engine MASCOT v 2.0(Matrix Science).

Isolation of circulating GPBP from human plasma—Ten milliliters ofplasmapheresis from Goodpasture patients were applied to aSepharose-CNBr (Sigma) column (1 ml bed) containing 5 mg of covalentlybound αGPBPr. The column was washed with 20 ml of TBS containing 0.05%Tween 20 (TBST) and eluted with Gentle-Immunopure elution buffer(Pierce). Eluted material was dialyzed against TBS, concentrated with aMicrocon YM-3 (Millipore) and further analyzed by Western blot usingαGPBPab.

Estimation of circulating GPBP levels—Individual wells of microtiterplates were coated overnight with αGPBPr (2 μg/ml in TBS) and furtherincubated with blocking buffer (3% BSA in PBS) for 2 h. Recombinant GPBPand serum samples were diluted in bovine foetal serum and incubated induplicate for 2 h. Plates were then incubated for 1 h each with αGPBPab(1:5,000 in TBS) and with anti-chicken HRP-conjugated (1:20,000 in TBS).All the steps except coating (4° C.) were at RT and wells were washedextensively with TBST between steps. Finally, detection was done usingAmplex UltraRed reagent (Invitrogen) with an excitation/emission maxima˜568/581 nm in a Victor 2 microtiter plate reader (PerkinElmer). Alinear range of the standard curve was found between 0.5 and 10 ng/ml ofrecombinant GPBP. We used Mann-Whitney test to assess differencesbetween series. A P value<0.05 was considered significant. Prism 4.0software (GraphPad Software, San Diego, Calif.) was used forcalculations.

SDS-PAGE and Western blot analysis—Were performed under reducingconditions following standard procedures and using chemiluminescence(Amersham Pharmacia Biotech) for antibody detection.

Results

COL4A3BP encodes for polypeptides of 77-, 91- and 120-kDa—To identifyGPBP and GPBPΔ26, we have used two different monoclonal antibodies: mAb14 previously reported to recognize GPBP and GPBPΔ26 (1), and mAb e26, anovel monoclonal antibody raised against the 26-residue Ser-rich regionexclusive for GPBP (FIG. 1A). Using GPBP deletion mutants and syntheticpeptides, we have mapped mAb 14 epitope to the FFAT motif and thus, thisantibody did not react with a GPBP mutant lacking the FFAT motif(GPBP_(ΔFFAT)) (FIG. 9).

Western blot analysis of cell extracts revealed that mAb 14 mainlyrecognized a single polypeptide with an apparent molecular weight(M_(r)) of ˜77-kDa (¹) whereas mAb e26 reacted with two polypeptides of˜91- and 120-kDa M_(r) (FIG. 1B). Minor and variable reactivity was alsoobserved towards polypeptides of ˜77-, 60-, 50- and 32-kDa with mAb e26and against polypeptides of ˜91- and 120-kDa with mAb 14 (not shown). Wefound similar reactive molecular species in a number of cultured humancells including HEK 293 (FIG. 1B), human fibroblasts, HeLa, hTERT-RPEand hTERT-BJ1 cells (not shown).

To further characterize COL4A3BP products, we compared expression ofnative and recombinant mRNAs (FIG. 1C). For these purposes, pc-n4′, aconstruct bearing the 5′UTR and coding sequence of COL4A3BP (1, 17), wasused in transient gene expression assays in cultured cells. Theexpression of pc-n4′ yielded three polypeptides of ˜77-, 91- and 120-kDawhich were detected by mAb e26. In contrast, only the ˜77- and 91-kDapolypeptides were significantly reactive with mAb 14. Strikingly, themost prominent mAb e26-reactive polypeptide in the recombinant lysates(77-kDa), representing the previously reported mRNA product (1), did nothave a significant native counterpart. We also observed that mAb 14reacted comparatively stronger with the 91- than with 120-kDarecombinant polypeptides.

To further determine the origin of native polypeptides, we used smallinterfering RNAs (siRNAs) specific for COL4A3BP (FIG. 1D). Theexpression of all three native polypeptides was reduced when expressingthese siRNAs; however, siRNA specific for both GPBP and GPBPΔ26/CERTwere more efficient at reducing the expression of 77-kDa polypeptidewhereas GPBP-specific siRNA reduced more effectively the expression of91- and 120-kDa polypeptides (compare pSi-GPBP/GPBPΔ26-3 and psiGPBP-1).Collectively, our data suggested that major cellular products ofCOL4A3BP included GPBPΔ26/CERT (77-kDa) and the previously unrecognizedGPBP isoforms of 91- and 120-kDa, the later likely bearing a modifiedFFAT motif that prevented consistent mAb 14 binding. The reduction inthe cellular levels of 77-kDa polypeptide when using GPBP-specificsiRNAs requires further investigation since this polypeptide displayedno significant reactivity with mAb e26 (FIG. 1B).

Major cellular GPBP isoforms result from noncanonical mRNA translationinitiation—To further define the origin of cellular GPBP isoforms, weproduced (pc-n4′)-derived constructs expressing mRNA mutants consistingof 5′UTR-deletion or iMet to Gly substitution (FIG. 2A) and these wereused in protein expression assays (FIG. 2B). In cells, the constructrepresenting 5′UTR-deleted mRNA (pc-GPBP-Met) produced only the 77-kDapolypeptide and the constructs representing the iMet to Gly substitution(pc-n4′-Mmut) expressed only the 91- and 120-kDa polypeptides (FIG. 2B,ex vivo). However, in a cell-free translation system, pc-GPBP-Met alsoexpressed 77-kDa GPBP polypeptide but pc-n4′Mmut yielded only the 91-kDapolypeptide and no significant expression of 120-kDa polypeptide wasobserved (FIG. 2B, in vitro). These data indicated that GPBP mRNAcontained a noncanonical translation initiation site(s) in the 5′UTRthat accounted for polypeptides of 91- and 120-kDa whereas the 77-kDapolypeptide was the product of canonical translation initiation.Moreover, our data also suggested that the 91-kDa was the primaryproduct of noncanonical translation initiation and the 120-kDapolypeptide represented a posttranslational derived product that couldnot be expressed in a cell-free system devoid of cellular membranes.

To characterize further noncanonical translation initiation, thepreviously recognized (1) ORF present in the 5′UTR of the GPBP mRNA(FIG. 2C) was interrupted by introducing a stop codon at individualpositions in pc-n4′Mmut and cellular protein expression assessed byWestern blot (FIG. 2D). The construct bearing a stop codon at −83(originally ACG, threonine) did not express the 91- and 120-kDapolypeptides, but the construct with the stop codon at −84 (originallyGCG, alanine) expressed the two polypeptides mapping the alternativetranslation start site to codon-83 (boxed Thr in FIG. 2C). The sameconclusion was obtained when we assayed the −83 stop-mutant of pc-n4′(FIG. 2D).

To confirm that noncanonical translation initiation also accounted forendogenous GPBP polypeptides of 91- and 120-kDa, a human F(ab)₂ fragment(Ab 24) specifically reacting with a synthetic peptide representing thepredicted ATR (shaded sequence in FIG. 2C) was used for Western blotanalysis of partially purified GPBP polypeptides (FIG. 2E). As expected,Ab 24 specifically reacted with two polypeptides of 91- and 120-kDawhich were also recognized by mAb e26, suggesting that native GPBPpolypeptides contained the ATR characteristic of noncanonicaltranslation products.

The 91- and 120-kDa GPBP isoforms are insoluble membrane-boundpolypeptides—GPBP isoform of 91-kDa was predicted to be non-classicalsecreted proteins when analyzed with SecretomeP 2.0 Server (18,http://www.cbs.dtu.dk/services/SecretomeP/) and to localize inmitochondria (60.9%), nucleus (26.1%), cytoskeleton (8.7%) and vesiclesof secretory system (4.3%) when analyzed with PSORT II Prediction(http://psort.ims.u-tokyo.acjp/form2.html). Thus, these theoreticalconsiderations suggested that GPBP isoforms resulting from noncanonicaltranslation initiation were noncytosolic polypeptides that entered intocellular organelles including the secretory pathway.

To assess these predictions, intact living cells were incubated withαGPBP and analyzed by direct immunofluorescence and flow cytometry forantibody binding detection (FIGS. 3A and 3B). Interestingly, αGPBP boundto living cells in a specific manner since binding of the antibodies wasefficiently abolished by a synthetic peptide representing GPBP(GPBPpep1) but not by an unrelated polypeptide (Contpep). These datasuggested that cellular GPBP isoforms were present in the externalsurface of the plasma membrane.

To further characterize the intracellular distribution of GPBP, cellswere disrupted and subjected to subcellular fractionation and Westernblot analysis (FIG. 3C). Consistent with predictions, GPBP isoforms of91- and 120-kDa were not detected as soluble materials but rather theywere found mainly associated with mitochondrial-lysosomal and microsomalfractions. It remained to be determined whether the presence of GPBP inthe nuclear fraction indeed reflected nuclear expression of theseproteins or rather unbroken cells and/or mitochondria contaminating thisfraction. In contrast, a polypeptide of ˜77-kDa which reacted with mAb14 and showed no significant reactivity with mAb e26 was exclusivelydetected as soluble after sample centrifugation at 150,000×g for 1 h(cytosol).

These data suggested that native GPBP polypeptides of 91- and 120-kDawere expressed insoluble associated with cellular membranes whereasnative GPBPΔ26/CERT polypeptide of 77-kDa was expressed soluble in thecytoplasm.

The 77-kDa GPBP is a soluble extracellular protein which interacts withtype IV collagen—Previous reports suggested that 77-kDa GPBP interactswith type IV collagen (1, 3, 4). This was further assessed by ex vivocross-linking and FLAG-immunoprecipitation of cells expressing or notexpressing BM40-FLAG-α3(IV)NC1, a recombinant exportable form of thehuman α3(IV)NC1 (16), followed by SDS-PAGE analysis ofimmunoprecipitates (FIG. 4A). FLAG-specific antibodies efficientlyprecipitated FLAG-α3(IV)NC1 and a 77-kDa polypeptide representing eitherGPBP or GPBPΔ26/CERT (²) (Western) along with Grp78 and Grp94(Coomassie), two ER resident chaperones implicated in protein foldingand ER homeostasis maintenance (19, 20). To further determine that GPBPindeed interacted with FLAG-α3(IV) in the ER, cells expressing or notexpressing BM40-FLAG-α3(IV)NC1 were transfected with pc-n4′ andsimilarly analyzed (FIG. 4B). FLAG antibodies efficiently precipitated77-kDa GPBP from cells expressing FLAG-α3(IV)NC1 but not from controlcells, suggesting that 77-kDa GPBP isoform enters into the secretorypathway and interacts with FLAG-α3(IV)NC1.

Primary structure analysis predicted a cytoplasmic localization for77-kDa GPBP polypeptide (unpublished observations). However, in vitro(1, 3), ex vivo (FIG. 4) and in vivo (4) studies suggested that 77-kDaGPBP isoform binds and phosphorylates type IV collagen. Furthermore,although recombinant expression studies revealed that the 77-kDa GPBPpolypeptide was the most prominent polypeptide, no significant levels ofthe native counterpart were detected within the cells (FIG. 1).Collectively, these observations suggested that canonical GPBP was acytosolic polypeptide subjected to a nonclassical secretion.

To explore whether GPBP is secreted, we first expressed FLAG-tagged GPBPin HeLa cells and used FLAG-specific antibodies to analyze intracellularrecombinant protein distribution (FIG. 5A). FLAG-GPBP co-localizedextensively with calregulin, an ER resident protein, suggesting that, asdescribed for GPBPΔ26/CERT (21, 22), FLAG-GPBP bound to the ER throughFFAT-VAP interaction. Consequently, we expressed and similarly analyzedFLAG-GPBP_(ΔFFAT), a FLAG-GPBP variant devoid of FFAT motif. Deletion ofFFAT motif prevented distribution of GPBP to the ER as the protein wasfound extensively co-localizing with golgin-97, a Golgi apparatusresident protein (FIG. 5A). Identical conclusions were obtained when thestudies were conducted in HEK 293 cells (not shown). Our data wereconsistent with the notion that recombinant GPBP was a cytosolic proteinbound to VAP through the FFAT motif for its exportation and only whenFFAT-interaction was impaired, the protein had the potential toassociate with Golgi apparatus. This was explored by expressingFLAG-GPBP or FLAG-GPBP_(ΔFFAT) in cultured cells and the subsequentanalysis of culture media by immunoprecipitation and Western blotanalysis (FIG. 5B). Interestingly, FLAG-specific antibodies efficientlyimmunoprecipitated recombinant protein from the media of culturesexpressing FLAG-GPBP but not from the media of cells expressingFLAG-GPBP_(ΔFFAT), revealing that FFAT-mediated binding to the ER isessential for 77-kDa GPBP secretion.

GPBPΔ26/CERT also binds to the ER in a FFAT-dependent manner (21, 22);however, we found GPBPΔ26/CERT in the cytosol and 77-kDa GPBP in theextracellular compartment, supporting that the Ser-rich 26-residueregion exclusive to GPBP is also critical for GPBP secretion. This wassimilarly explored in cultures expressing FLAG-tagged 77-kDa GPBP orGPBPΔ26/CERT (FIG. 5C). As expected, the presence of the 26-residue Serrich region was critical for protein secretion given that FLAG-GPBPΔ26was not significantly expressed in the culture media.

The 91-kDa GPBP regulates the levels of 77-kDa GPBP in the extracellularcompartment—The evidence supports that both the 77- and 91-kDa GPBPisoforms enter into the secretory pathway but whereas the 91-kDa remainsassociated to membranes, the 77-kDa GPBP is soluble in the extracellularcompartment. We have explored whether 91-kDa GPBP regulates theextracellular levels of 77-kDa GPBP. This was accomplished byrecombinant expression of FLAG-GPBP in individual cell lines expressingrecombinant 91-kDa GPBP to a different levels (FIG. 6A) followed byFLAG-immunoprecipitation of the corresponding cultured media andanalysis of immunoprecipitates by Western blot (FIG. 6B). Interestingly,increased expression of recombinant 91-kDa GPBP associated withincreased levels of FLAG-GPBP in the culture media, suggesting that91-kDa GPBP induced the secretion of 77-kDa GPBP to the extracellularcompartment.

The 77-kDa GPBP is not sensitive to cell treatment withsphingomyelinase—Recombinant expression studies also showed that 77-kDaGPBP was a cytosolic polypeptide associated with ER that underwenttranslocation to the Golgi apparatus when FFAT motif was mutated (FIG.5A). Consequently, we asked whether 77-kDa GPBP underwentdephosphorylation and translocation to the Golgi apparatus in responseto sphingomyelinase cell treatment as previously reported forGPBPΔ26/CERT (5). For these studies, cells expressing FLAG-tagged GPBPor GPBPΔ26/CERT were treated with Bacillus cereus sphingomyelinase(bSMase) and intracellular proteins of interest were analyzed byFLAG-immunoprecipitation and Western blot (FIG. 7A). As previously noted(1, 5), both recombinant proteins were phosphorylated and treatment witha general phosphatase (λPPase) reduced their M_(r) to a similar extent(top and bottom arrows). However, sphingomyelinase cell treatment haddifferent consequences for each recombinant protein; whereasFLAG-GPBPΔ26/CERT shifted to a lower M_(r) (top and middle arrows), nosignificant M_(r) shift was observed for FLAG-GPBP. This suggested thatthe reduction in the cellular levels of sphingomyelin caused bysphingomyelinase treatment induced the dephosphorylation ofFLAG-GPBPΔ26/CERT but did not affect significantly the phosphorylationstate of FLAG-GPBP. As expected, immunofluorescence analysis of thecells revealed that sphingomyelinase treatment promoted translocation ofFLAG-GPBPΔ26/CERT to the Golgi apparatus without altering significantlythe intracellular distribution of FLAG-GPBP (FIG. 7B).

Circulating levels of 77-kDa GPBP are upregulated in Goodpasturepatients and in animal models of immune complex-mediatedglomerulonephritis—Evidence suggested that 77-kDa GPBP was secreted as asoluble protein in vivo was first investigated by immunoaffinitychromatography to isolate circulating human 77-kDa GPBP (FIG. 8A). Weused plasmapheresis obtained by standard therapeutic procedures fromGoodpasture patients, which were predicted to express higher levels ofGPBP (3). As expected, we identified a single polypeptide of 77-kDa inthe material eluted from the affinity column which reacted with theGPBP-specific antibodies, suggesting that 77-kDa GPBP is secreted invivo and is a component of the human plasma. To both validate affinitypurification and determine the levels of 77-kDa GPBP in a more precisemanner, we developed an ELISA employing the same antibodies which wereused in affinity chromatography to capture and detect human recombinantGPBP (FIG. 8B). We used this ELISA to estimate circulating 77-kDa GPBPlevels in samples representing control and antibody-mediatedglomerulonephritis (FIGS. 8C, D). The ELISA displayed a linear rangebetween 0.5 ng and 10 ng/ml when measuring recombinant GPBP (FIG. 8B)and detected comparatively more circulating 77-kDa GPBP in Goodpasturepatients than in control individuals (FIG. 8C). We obtained similarresults when comparing young (4-month) and aged (7-month) NZW mice (FIG.8D), a mouse strain that develops GPBP-dependent IgA immunecomplex-mediated glomerulonephritis and lupus-prone autoantibodyproduction commencing at 7 months of age (4).

Discussion

Here we have obtained compelling evidence that the mRNA of GPBPundergoes canonical (AUG) and noncanonical (ACG) translation initiationto generate two primary polypeptides of 77- and 91-kDa, respectively.The results from the present study also support that both products enterthe secretory pathway. However, whereas the 77-kDa reaches theextracellular compartment and exists in a soluble immunoprecipitableform, the 91- and its derived 120-kDa polypeptides remain mainlyinsoluble, associated with cellular membranes. The use of translationinitiation at ACG and noncanonical translation initiation to directproteins to alternative cell compartments has been described for otherhuman genes (23, 24). Based on previous evidence (21, 22), it isexpected that FFAT-mediated GPBP binding to the ER (FIG. 5) occursthrough VAP and therefore that FFAT-VAP interaction mediates molecularmechanisms underlying

GPBP translocation into the ER. Furthermore, we also show that thepreviously reported alternatively-spliced GPBPΔ26/CERT is a GPBP variantthat remains mainly soluble in the cytoplasm. Thus, our data support thenotion that mRNA alternative translation initiation and exon splicingare strategies to direct GPBP to multiple locations including thecytosol, secretory pathway, plasma membrane and extracellularcompartment. Moreover, previous observations have localized GPBP to thenucleus in human spermatogonium (1) and in the mitochondria and lysosomeof rat liver (unpublished observations), suggesting that thedistribution of GPBP is virtually ubiquitous and therefore, itsbiological program is expected to be exerted in several compartments.

A human GPBP cDNA from pulmonary artery endothelial cell has beenreported (GenBank accession number AK096854). Interestingly, AK096854bears an alternative canonical translation initiation site (iMet) thatextends the ORF of the 91-kDa polypeptide upstream by 45 residues. Wehave not found evidence for AK096854 mRNA expression in HEK 293 cells,nor in a number of other human tissues including liver, kidney, brain,muscle, pancreas, keratinocytes, lymphocytes and HeLa cells (not shown).Nevertheless, the existence of GPBP isoforms produced by canonical mRNAtranslation initiation (i.e. AK096854) with a M_(r) similar to that ofthe noncanonical translation initiation products reported here cannot beexcluded.

Primary structure analysis predicts that noncanonically translated GPBPproducts enter into the secretory pathway. Several observations supportthese predictions, namely: 1) noncanonical GPBP isoforms are molecularspecies associated with cellular membranes (FIG. 3); 2) noncanonicalGPBP isoforms are the predominant GPBP species in the cell (FIG. 1) andGPBP-specific antibodies bound to the external surface of intact livingcells (FIG. 3); 3) 120-kDa polypeptide is not expressed from the mRNAwhen translation occurs in a cell-free system devoid of cellularmembranes (FIGS. 2); and 4) 91-kDa GPBP isoform regulates the levels ofthe 77-kDa GPBP at the extracellular compartment (FIG. 6). Takentogether, these observations support the notion that the 91-kDapolypeptide is the primary product of noncanonical translationinitiation. This isoform enters into the secretory pathway whereundergoes covalent modification to yield the 120-kDa polypeptide andremains bound to membranes reaching the external surface of the plasmamembrane. The mechanism by which 91-kDa GPBP regulates the extracellularlevels of 77-kDa GPBP remains unknown.

We have observed that when expression is abnormally elevated (i.e.transient gene expression), GPBP polypeptides accumulate in the cytosol(FIG. 10), revealing that GPBP transportation into the ER is a saturableprocess. Interestingly, under these expression conditions, mAb e26displayed more reactivity for the cytosolic 77-kDa polypeptide than forthis isoform when residing in the extracellular compartment (FIGS. 10and 11). Moreover, mAb 14 reacted comparatively more with recombinantthan with native 91-kDa GPBP and did not react significantly with nativeor recombinant 120-kDa product (FIG. 1). All these observations suggestthat the 26-residue Ser-rich region (mAb e26) and the FFAT motif (mAb14) are subjected to covalent modifications in the secretory pathway.These data also imply that under specific regulatory (physiological orpathological) circumstances GPBP can be expressed as solublepolypeptides in the cytosol. Finally, it remains to be determinedwhether 91-kDa GPBPΔ26/CERT is expressed endogenously and whetherGPBPΔ26/CERT can be transported into the ER without undergoingsecretion.

The expression levels of cytosolic 77-kDa polypeptide representingGPBPΔ26/CERT were significantly reduced in cells expressingGPBP-specific siRNA (FIG. 1D). This suggests that either siRNA is alsotargeting the pre-mRNA or that the mRNA of GPBP is to some extent aprecursor of GPBPΔ26 mRNA. We have found that cells expressingrecombinant GPBP also expressed limited amounts of recombinantGPBPΔ26/CERT (unpublished observations). This reveals that mature GPBPmRNA is subjected to a nonclassical processing, similarly to thatreported for XBP1 in response to ER stress signals (25). Alternatively,GPBP species bearing covalently modified 26-residue Ser-rich regionwhich co-migrate with GPBPΔ26/CERT could also account for thisobservation.

Several lines of evidence support that GPBP regulates protein folding inthe ER and supramolecular organization in the extracellular compartmentrather than inter-organelle ceramide traffic in the cytosol: 1) The77-kDa GPBP is a nonconventional Ser/Thr kinase that binds andphosphorylates the α3(IV)NC1 domain at sites (1) that are alsophosphorylated in vivo (26); 2) The 77-kDa GPBP is mainly found in theextracellular compartment both soluble (FIG. 5 and FIG. 8) or associatedwith GBM collagen (4), and is not expressed at significant levels in thecytosol of cultured cells (FIGS. 1 and 3); 3) Cellular GPBP isoformslocalize at the external surface of the plasma membrane (FIG. 3); 4) The91-kDa GPBP isoform is associated with cellular membranes (FIG. 3) andregulates the extracellular levels of the 77-kDa GPBP isoform (FIG. 6);5) The α3(IV)NC1 domain undergoes unique structural diversification andat least two distinct conformational isoforms (conformers) assemble inbasement membranes (27); 6) An increased expression of the 77-kDa GPBPperturbs the quaternary structure of type IV collagen, suggesting thatthe elevated GPBP levels interferes with the conformationaldiversification program (tertiary structure) of the α3(IV)NC1 domain(4); 7) Increased serum levels of GPBP correlates with type IV-collagenbased glomerulonephritis (FIG. 8); 8) The FFAT motif is a structuralrequirement for 77-kDa GPBP secretion (FIG. 5) and VAP is critical formaintaining the homeostasis for adequate protein folding in the ER (10);9) Grp78 and Grp94, chaperones which reside in the ER and regulatecellular response to protein misfolding (18, 19), are associated withFLAG-α3(IV) and 77-kDa GPBP (FIG. 4); 10) Increased COL4A3BP expressionhas been found to mediate resistance of cancer cells to chemotherapeuticagents that induce protein misfolding and ER stress-mediated cell death(28); 11) Treatment of cells with sphingomyelinase does not inducedephosphorylation nor does it alter intracellular distribution of 77-kDaGPBP (FIG. 7); 12) Protein kinase D phosphorylates GPBP but not to thesame extent as GPBPΔ26/CERT (6); 13) Knock-down and rescue experimentsreveal that GPBP and GPBPΔ26/CERT exert different biological functionsduring embryogenesis in Zebra fish (29); and 14) GPBP interacts withproteins RTN3 and RTN4 which are anchored from the luminal/extracellularside to the membranes in the secretory pathway (30).

GPBP lacking the 26-residue Ser-rich region also binds to VAP (21, 22);however, ceramide uptake follows binding to VAP and subsequently, theprotein departs to the Golgi apparatus where ceramide is released andprotein exocytosis induced (6, 14). Therefore, phosphate transfer andceramide trafficking may be molecular strategies through which COL4A3BPregulates protein secretion (i.e. type IV collagen). Consistent withthis, it has been shown that VAP is also critical for regulating proteincargo transport to the plasma membrane (11).

Various lines of evidence support that COL4A3BP is an attractive targetfor strategies to diagnose and treat antibody-mediated disorders (3, 4),inflammation (15), ER stress-mediated diseases (10) and drug resistantcancer (28). However, observations supporting these conclusions may nowneed to be re-interpreted since many have been obtained using tools(i.e. siRNA or antibodies) which failed to discriminate betweendifferent gene products (i.e. GPBP and GPBPΔ26/CERT), that are expressedat distinct cell compartments, and are differentially regulated inresponse to stimuli (3). Therefore, the present study makes an importantcontribution to this understanding by clarifying the mechanisms by whichvarious isoforms of GPBP are generated within the cells.

Furthermore, by identifying circulating human 77-kDa GPBP, we providecompelling evidence that GPBP secretion is also biologically relevant invivo. The finding that the levels of circulating 77-kDa GPBP correlatewith GPBP glomerular expression and pathogenesis in mouse models ofimmune complex-mediated glomerulonephritis suggests that serologicaldetermination of GPBP is relevant in a clinical setting. Consistent withthis, present studies demonstrating upregulation of circulating GPBP inGoodpasture patients support these conclusions and substantiate previousobservations that GPBP is overexpressed in these patients (3, 31).

These and previous findings support that GPBP promotes type IV collagensecretion and supramolecular organization. Accordingly, GPBP is criticalfor adequate GBM assembly and abnormal GPBP accumulation induces GBMdisruption and deposits of IgA immune complexes (4). To our knowledge,increased GPBP expression, GBM dissociation and deposits of immunecomplexes are novel mechanisms underlying renal disease. Whether similarmechanisms operate in human pathogenesis remains to be determined;however, ultrastructural evidence for GBM disruption and accumulation ofelectron-dense material has been reported in patients undergoing IgAnephropathy and lupus nephritis (32, 33). Moreover, increased GPBPexpression could reduce the reinforcement of the quaternary structure oftype IV collagen, thereby facilitating epitope exposure, immune systemactivation and autoantibody binding in Goodpasture disease (34).Consistent with the later hypothesis, Goodpasture patients presentincreased levels of circulating GPBP supporting previous observationsthat GPBP expression is upregulated in Goodpasture tissues (3, 31). GPBPis a circulating molecule and GBM a principal component of theglomerular filtration barrier; therefore, pathogenic GPBP accumulationin the glomerulus could result from local production but also from thesequestration of circulating GPBP produced elsewhere. The localoverproduction could account for primary antibody-mediatedglomerulonephritis whereas increased circulating levels may inducesecondary forms of this pathology and perhaps are responsible fordisease recurrence upon renal transplantation. Consequently,quantification of the levels of circulating GPBP might be useful indiscriminating primary from secondary antibody-mediatedglomerulonephritis and for the clinical monitoring of renaltransplantation.

References for Example 1

-   1. Raya, A., Revert, F., Navarro, S., and Saus, J. (1999) J. Biol.    Chem. 274, 12642-12649-   2. Hudson, B. G., Tryggvason, K., Sundaramoorthy, M., and    Neilson, E. G. (2003) N Engl. J. Med. 348, 2543-2556-   3. Raya, A., Revert-Ros, F., Martínez-Martínez, P., Navarro, S.,    Roselló, E., Vieites, B., Granero, F., Forteza, J., and    Saus, J. (2000) J. Biol. Chem. 275, 40392-40399-   4. Revert, F., Merino, R., Monteagudo, C., Macías, J., Peydró, A.,    Alcácer, J., Muniesa, P., Marquina, R., Blanco, M., Iglesias, M.,    Revert-Ros, F., Merino, J., and Saus, J. (2007) Am. J. Pathol. 171,    1419-1430-   5. Kumagai, K., Kawano, M., Shinkai-Ouchi, F., Nishijima, M., and    Hanada, K. (2007) J. Biol. Chem. 282, 17758-17766-   6. Fugmann, T., Hausser, A., Schöffler, P., Schmid, S., Pfizenmaier,    K., and Olayioye, M. A. (2007) J. Cell Biol. 178, 15-22-   7. Lemmon, M. A., and Ferguson, K. M. (2000) Biochem J. 350, 1-18-   8. Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G.,    Downes, C. P., and Alessi, D. R. (2000) Biochem. J. 351, 19-31-   9. Loewen, C. J. R., Roy, A., and Levine, T. P. (2003) EMBO J. 22,    2025-2035-   10. Kanekura, K., Nishimoto, I., Aiso, S., and    Matsuoka, M. (2006) J. Biol. Chem. 281, 30223-30233-   11. Wyles, J. P., McMaster, C. R., and Ridgway, N. D. (2002) J.    Biol. Chem. 277, 29908-29918-   12. Soccio, R. E. and Breslow, J. L. (2003) J. Biol. Chem. 278,    22183-22186-   13. Alpy, F., and Tomasetto, C. (2005) J. Cell Sci. 118, 2791-2801-   14. Hanada, K., Kumagai, K., Yasuda, S., Miura, Y., Kawano, M.,    Fukasawa, M., and Nishijima, M. (2003) Nature 426, 803-809-   15. Lamour, N. F., Stahelin, R. V., Wijesinghe, D. S., Maceyka, M.,    Wang, E., Allegood, J. C., Merrill, A. H. Jr., Cho, W.,    Chalfant, C. E. (2007) J. Lipid Res. 48, 1293-1304-   16. Netzer, K. 0., Leinonen, A., Boutaud, A., Borza, D. B., Todd,    P., Gunwar, S., Langeveld, J. P., and Hudson, B. G. (1999) J. Biol.    Chem. 274, 11267-11274-   17. Granero, F., Revert, F., Revert-Ros, F., Lainez, S.,    Martínez-Martínez, P., and Saus, J. (2005) FEBS J. 272, 5291-5305-   18. Bendtsen, D. J., Jensen, J. L., Blom, N., von Heijne, G., and    Brunak, S. (2004) Protein Eng. Des. Sel. 17, 349-356-   19. Yang, Y., and Li, Z. (2005) Mol. Cells. 20, 173-182-   20. Ni, M., and Lee, A. S. (2007) FEBS Lett. 581, 3641-3651-   21. Perry, R. J., and Ridgway, N. D. (2006) Mol. Biol. Cell. 17,    2604-2616-   22. Kawano, M., Kumagai, K., Nishijima, M., and Hanada, K. (2006) J.    Biol. Chem. 281, 30279-30288-   23. Peabody, D. S. (1989) J. Biol. Chem. 264, 5031-5035-   24. Touriol, C., Bornes, S., Bonnal, S., Audigier, S., Prats, H.,    Prats, A. C., and Vagner, S. (2003) Biol. Cell. 95, 169-178-   25. Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and    Mori, K. (2001) Cell 107, 881-891-   26. Revert, F., Penadés, J. R., Plana, M., Bernal, D., Johansson,    C., Itarte, E., Cervera, J., Wieslander, J., Quinones, S., and    Saus, J. (1995) J. Biol. Chem. 270, 13254-13261-   27. Calvete, J. J., Revert, F., Blanco, M., Cervera, J., Tárrega,    C., Sanz, L., Revert-Ros, F., Granero, F., Pérez-Payá, E.,    Hudson, B. G., and Saus, J. (2006) Proteomics 6, S237-S244-   28. Swanton, C., Marani, M., Pardo, O., Warne, P. H., Kelly, G.,    Sahai, E., Elustondo, F., Chang, J., Temple, J., Ahmed, A. A.,    Brenton, J. D., Downward, J., and Nicke, B. (2007) Cancer Cell 11,    498-512-   29. Granero-Moltó, F., Sarmah, S., O'Rear, L., Spagnoli, A.,    Abrahamson, D., Saus, J., Hudson, B. G., and Knapik, E. W. (2008) J.    Biol. Chem. Apr. 18. [Epub ahead of print]-   30. Rual, J. F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T.,    Dricot, A., Li, N., Berriz, G. F., Gibbons, F. D., Dreze, M.,    Ayivi-Guedehoussou, N., Klitgord, N., Simon, C., Boxem, M.,    Milstein, S., Rosenberg, J., Goldberg, D. S., Zhang, L. V., Wong, S.    L., Franklin, G., Li, S., Albala, J. S., Lim, J., Fraughton, C.,    Llamosas, E., Cevik, S., Bex, C., Lamesch, P., Sikorski, R. S.,    Vandenhaute, J., Zoghbi, H. Y., Smolyar, A., Bosak, S., Sequerra,    R., Doucette-Stamm, L., Cusick, M. E., Hill, D. E., Roth, F. P., and    Vidal, M. (2005) Nature 437, 1173-1178-   31. Saus, J (2002) “Methods and reagents for treating autoimmune    disorders” Utility Patent Application serial n PCT/EP02/01010.    Publication no WO 02/061430-   32. Haas M. “IgA Nephropathy and Henoch-Schönlein Purpura    Nephritis”. Heptinstall's Pathology of the Kidney. Edited by    Jennette J C, Olson J L, Schwartz M M, Silva F G. Philadelphia,    Lippincott Williams & Wilkins Publishers 2007, pp. 423-486-   33. Balow, J. E., Boumpas, D. T., and Austin III, H. A. “Systemic    Lupus Erythematosus and the kidney”. Systemic Lupus Erythematosus.    Edited by Lahita RG. San Diego, Academic Press, 1999, pp. 657-685-   34. Borza, D. B., Bondar, O., Colon, S., Todd, P., Sado, Y.,    Neilson, E. G., and Hudson, B. G. (2005) J. Biol. Chem. 280,    27147-27154

Footnotes

(1) The 77-kDa polypeptide can be resolved as a doublet representingphosphorylated (higher) and dephosphorylated (lower) versions ofGPBPΔ26/CERT (5)(2) Secretion of 77-kDa GPBP associated with loss of reactivity with mAbe26 (FIG. 11), excluding the use of this antibody to estimate the levelsof native 77-kDa GPBP in the secretory pathway.

Abbreviations

The abbreviations used are: α3(IV)NC1, the NC1 domain of the α3 chain oftype IV collagen; ATR, alternative translated region; CERT and CERT_(L),short and large isoforms of the ceramide transfer protein; COL4A3BP, thegene encoding for GPBP (CERT_(L)) and GPBPΔ26 (CERT) which was namedcollagen IV α3-binding protein; EDTA, ethylenediaminetetraacetic acid;ER, endoplasmic reticulum; FFAT, two phenylalalines in an acidic track;GBM, glomerular basement membrane; GPBP and GPBPΔ26, large and shortalternatively spliced variants of the Goodpasture antigen-bindingprotein; HRP, horseradish peroxidase; mAb, monoclonal antibody; NC1,noncollagenous-1 domain; ORF, open reading frame; NZW, new Zealandwhite; PBS, phosphate buffered-saline; PH, pleckstrin homology; RT, roomtemperature; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; START, steroidogenic acute regulatory related lipidtransfer; UTR, untranslated region; VAP, vesicle associated membraneprotein-associated protein.

Example 2 Identification and Isolation of GPBP from Human Plasma

Here we used classical chemical procedures for protein fractionation ofhuman plasma and identified multiple tertiary and quaternary GPBPstructures circulating in human plasma. The data also show that 77-kDaGPBP and derived species of lower MW are the major GPBP circulatingisoform(s) as determined by reconstitution of plasma conditions fromisolated partially purified-GPBP quaternary structures.

Materials and Methods

GPBP was purified from 50 ml of frozen control plasma using acombination of salting-out precipitation, ion exchange chromatographyand gel filtration.

Proteins precipitated by freezing were first removed by plasmacentrifugation at 8200×g for 10 min at 4° C. Since the specificproperties for purification of plasma GPBP are not known, proteins weresequentially precipitated from the original sample with growing(NH₄)₂SO₄ saturations (20%, 40%, 60% and 80%). Sequential precipitationswere performed by centrifugation at 8200×g for 10 min at 4° C. andprecipitates were dissolved in 5 ml of 50 mM Tris-HCl, pH 7.5. Proteinmixtures were desalted by dialysis against 50 mM Tris-HCl, pH 7.5 usingmembrane bags with 3.5-kDa cut-off The final supernatant of (NH₄)₂SO₄precipitations was similarly dialyzed and further used for purificationas the final fraction yielded by the precipitation process.

The fractions rendered by the salting-out were subsequently analyzed byion exchange chromatography (IEC) using a HiTrap Q-sepharose anionexchange column. The column was first equilibrated with buffer A (50 mMTris/HCl, pH 7.5, 20 mM NaCl), further loaded with each individualsample and washed with 10 volumes of buffer A. Bound proteins wereeluted with a gradient from buffer A to buffer B (50 mM Tris/HCl, pH7.5, 1 M NaCl) and collected in 0.6 ml fractions. IEC fractionscontaining GPBP material were detected by Western blot withGPBP-specific biotinylated N27 monoclonal antibody. GPBP-containing IECfractions were pooled, concentrated to 0.5 ml, and subsequentlysubjected to gel filtration chromatography with Superdex™ 200 10/300 CL.In this process, the column was first equilibrated with TBS (50 mMTris/HCl pH 7.5, 150 mM NaCl), the sample was injected into the columnand proteins separated by size. The gel filtration fractions wereanalysed by Western blot for detection of GPBP material withbiotinylated N27 monoclonal antibody. The fractions containing GPBP werepooled, precipitated with 80% acetone and resuspended in 50 mM Tris-HClpH 7.5, 8M urea. The resulting mixtures, each one corresponding to adifferent initial fraction rendered by sequential (NH₄)₂SO₄precipitation were pooled in equal proportions in order to faithfullyreconstitute the native plasma protein composition. A sample of thefinal pool was subjected to Western blot with HRP-labelled N27monoclonal antibody.

Results

In the resulting Western blot we observed major GPBP isoforms of 77-,70-, 66-, 58-, 56- and 53-kDa. There exist additional polypeptides notrepresented in significant amounts in the Western blot in FIG. 12 thatwere identified in Western blot analysis performed during thepurification process. These included polypeptides of approximately:368-kDa [20% (NH₄)₂SO₄], 40-, 110-, 120- and 311-kDa [40% (NH₄)₂SO₄],and 91-, 146-, 171- and 300-kDa polypeptides [60% (NH₄)₂SO₄] (data notshown). Finally, the size of each chromatographic peak in gel filtrationanalysis, which represented individual GPBP quaternary structures wasalso estimated. Specifically, we found GPBP aggregates of: 1400- and920-kDa in 20% (NH₄)₂SO₄precipitate; 310- and 145-kDa in 40% (NH₄)₂SO₄precipitate; 920-, 420-, 270-, and 125-kDa in 60% (NH₄)₂SO₄ precipitate;66-kDa in the 80% (NH₄)₂SO₄ precipitate; and, 91-kDa in a soluble format 80% (NH₄)₂SO₄ saturation.

Conclusions

-   -   1. There exist multiple circulating GPBP isoforms which are        assembled in a number of different quaternary structures.    -   2. The major circulating GPBP isoform includes the previously        recognized 77-kDa and derived polypeptides of lower M_(r).

Example 3 GPBP Isolation and Quantification from Human Urine

Here we demonstrate that GPBP is a normal component of the urine whichcan be both measured by simple immunological-based procedures (i.e.ELISA) and isolated by chemical and immunochemical procedures. Theevidence indicates that 91-kDa polypeptide and derived polypeptides arethe major urinary GPBP products.

Isolation of Urinary GPBP by Immunoaffinity Chromatography.

GPBP was extracted from urine of a control donor using Sepharose 4Bloaded with GPBP-specific rabbit polyclonal antibodies. The column-boundmaterial was eluted and analyzed by Western blot using GPBP-specificchicken polyclonal antibodies (FIG. 13). A number of polypeptidesdisplaying a broad range of MW were detected with GPBP-specificantibodies in the immunoaffinity purified sample. A 91-kDa polypeptide,along with other derived polypeptides of lower MW (46- and 50-kDa) [JuanSans, Fernando Revert and Francisco Revert-Ros “Novel Goodpastureantigen-binding protein isoforms and protein misfolded-mediateddisorders” PCT/EP04/01074 y WO 2004/070025], was found to be the mostabundant GPBP material in the human urine.

Specifically, two hundred and fifty milliliters of urine from a controldonor (previously cleared by centrifugation and neutralized with Tris),were loaded onto a 1 mL column of Sepharose 4B-conjugated with 200 μg ofrabbit polyclonal anti-GPBP antibodies. The column was washed with 30 mLof TBS and the bound material was eluted with Gentle Immunopure ElutionBuffer (Pierce). The material eluted was dialyzed against TBS andfurther analyzed by Western blot using GPBP-specific chicken polyclonalantibodies (αGPBPch) and HRP-labelled anti-chicken IgY (secondaryantibody) (FIG. 13). Antibody specificity was confirmed by staining acontrol lane loaded with the same material with secondary antibody(Cont). Bars and numbers or arrows and numbers indicate the position andsize (kDa) of MW standards (left) or GPBP polypeptides (right),respectively

Measurement of Urinary GPBP by ELISA

Since the concentration of protein in urine is low (normally lower than80 μg/mL), indirect ELISA was attempted with samples from seven donors.For these purposes, plates were coated with urine samples andimmunodetection performed using GPBP-specific chicken polyclonalantibodies and HRP-labelled anti-chicken IgY (secondary antibody). Astandard curve was similarly obtained using human recombinant GPBPdiluted in human urine. GPBP was detected in all donors and individualconcentrations were determined by subtracting the background (F.I.measured using unspecific IgY) in each case (FIG. 14). All donors showeddetectable levels of GPBP and donor 3 displayed an abnormally elevatedGPBP concentration in urine.

Specifically, recombinant GPBP diluted in urine and urine samples fromseven donors (1-7) were coated onto ELISA plates overnight at 4° C.Plates were blocked with 3% BSA in PBS and immunodetection performedwith GPBP-specific chicken polyclonal antibodies (αGPBPch) andHRP-labelled anti-chicken IgY (secondary antibody). Amplex UltraRedreagent (Invitrogen) was used for developing the plate. (FIG. 14) In A,is represented a scatter plot on a log-log scale of the indicatedconcentrations of GPBP versus fluorescence intensity (F.I.) expressed inarbitrary units (A.U.). In B, is represented the linear regression linecalculated with the indicated concentrations and their respective F.I.values plotted on linear scale, that was used to determine GPBP sampleconcentration in D. In C, is represented raw data obtained analyzingdonor samples with: secondary antibody (Cont), nonspecific chicken IgYand secondary antibody (IgY), or with αGPBPch and secondary antibody(αGPBPch). In D, the table shows corresponding transformed data usingthe curve obtained in B.

We obtained similar concentration values when GPBP was determined onTBS-diluted urine using the sandwich ELISA procedure used forserum/plasma samples (data not shown).

Urinary GPBP Isolation by Salt Precipitation and Ion ExchangeChromatography.

To validate immunoaffinity and ELISA procedures and to determine whichGPBP species increased in donor 3, we attempt GPBP purification fromthis urine using classical chemical purification procedures. Theseincluded, salt precipitation and double ion-exchange chromatography[carboxymethyl-cellulose (CM) and diethylaminoethyl-cellulose (DEAE)],and Western blot analysis of the different materials representing eachpurification step (FIG. 15). Western blot analysis using GPBP-specificchicken polyclonal antibodies revealed that most of GPBP material wasprecipitated by salt and did not bound to either CM or DEAE. A majorGPBP polypeptide of 91-kDa was detected along with significant amountsof GPBP polypeptide of 77-kDa and only traces of GPBP-relatedpolypeptides of 60- and 50-kDa.

To validate immunoaffinity and ELISA procedures and to determine whichGPBP species increased in donor 3, we attempted GPBP purification fromthis urine using classical chemical purification procedures. Fourhundred milliliters of urine cleared by centrifugation was brought to0.85 M NaCl overnight at 4° C., and subjected to centrifugation at10.000×g for 30 min at 4° C. A sample of the supernatant (Spt 0.85 MNaCl) was stored at 4° C. to be included in the subsequent analysis. Theresulting pellet was dissolved in 50 mM Tris pH 7.5, dialyzed againstthe same buffer, extracted with 0.7 mL of CM resin and unbound materialfurther extracted with 0.5 mL of DEAE resin. CM resin was eluted with 1MNaCl, 50 mM Tris pH 7.5 (CM, 1M NaCl), and DEAE resin was subsequentlyeluted with 0.35M NaCl, 50 mM Tris pH 7.5 (DEAE, 0.35M NaCl) and 1MNaCl, 50 mM Tris pH 7.5 (DEAE, 1M NaCl). Equivalent amounts of eachsample including the supernatant of the DEAE extraction (Spt CM/DEAE)were analyzed by Western blot with GPBP-specific chicken polyclonalantibodies and HRP-labelled anti-chicken IgY (αGPBPch). Nonspecificreactive polypeptides were identified by staining an in-parallelanalysis using only HRP-labelled anti-chicken IgY (Cont). Bars andnumbers or arrows and numbers indicate the position and size (kDa) of MWstandards (left) or polypeptides specifically reacting with anti-GPBPantibodies and that were detected only in SptCM/DEAE (right),respectively. (FIG. 15). Western blot analysis using GPBP-specificchicken polyclonal antibodies revealed that most of GPBP material wasprecipitated by salt and did not bound to either CM or DEAE. A majorGPBP polypeptide of 91-kDa was detected along with significant amountsof GPBP polypeptide of 77-kDa and only traces of GPBP-relatedpolypeptides of 60- and 50-kDa.

Conclusions

-   -   1) GPBP polypeptides can be isolated from urine either by        affinity chromatography or by salting-out precipitation followed        by ion-exchange chromatography.    -   2) GPBP levels in urine can be assessed either by indirect ELISA        or sandwich ELISA using specific anti-GPBP antibodies.    -   3) The major GPBP polypeptide found in urine displays 91-kDa.

Example 4 Production and Characterization of Monoclonal AntibodiesTargeting GPBP

Previously reported mAb14 and mAb e26 epitopes in GPBP are subjected toposttranslational modifications during secretion (Revert et al. 2008 J.Biol. Chem. 283:30246-55). Accordingly, these monoclonal antibodies didnot significantly react with circulating GPBP isoforms present in humanplasma. This recommended the use of polyclonal antibody-basedimmunological procedures for the isolation and estimation of GPBPcirculating levels in human plasma (see Example 1). Here we report theproduction and characterization of novel GPBP-specific monoclonalantibodies for immunological detection of GPBP in plasma which are morereliable than the polyclonal antibody-based strategy.

Propagation and cryopreservation of hybridomas producing new monoclonalantibodies against GPBP. Using indirect ELISA and recombinant GPBP madein yeast, we have obtained and isolated 28 independent hybridoma clones(N-1-N28) which produced anti-GPBP monoclonal antibodies. The cloneswere expanded in Dulbecco's Modified Eagle's Medium (DMEM) supplementedwith 20% fetal bovine serum (FBS), frozen in 10% DMSO in FBS and storedin liquid nitrogen. Before storage, 10 mL of culture medium from eachclone were collected stored at 4° C. with 0.01% sodium azide and usedfor further antibody characterization (see below).Western blot characterization of new monoclonal antibodies usingrecombinant and native GPBP isoforms expressed in HEK 293 cells. Theantibodies from each of the 28 hybridomas reacted with recombinant GPBP(25 ng) produced in E. coli (data not shown). Except for N20 and N21,all the rest of antibodies also reacted with intracellular recombinantGPBP (FIG. 16). Eleven monoclonal antibodies (N4, N5, N7, N11, N12, N13,N14, N22, N25, N27 and N28) recognized in a similar fashion both,intracellular and extracellular recombinant GPBP. Seven antibodies (N1,N6, N17, N18, N19, N24 and N26) target intracellular but notextracellular GPBP, while the remaining antibodies (N2, N3, N8, N9, N10,N15, N16 and N23) displayed relatively low reactivity with extracellularrecombinant GPBP (FIG. 16).

Using protein extracts from HEK 293 cells, we have determined that 18monoclonal antibodies [N2, N3, N4, N5, N7, N8, N9, N10, N11, N12, N13,N14, N15, N16, N22, N25, N27 (shown) and N28 (not shown)] recognizednative intracellular 77-kDa GPBP isoforms. Eleven out of these 18antibodies [N4, N5, N10, N11, N12, N13, N14, N16, N25, N27 (shown) notN28 (not shown)] also targeted a 45-kDa GPBP isoform previously reportedto exist in the cells [Juan Saus, Fernando Revert and FranciscoRevert-Ros “Novel Goodpasture antigen-binding protein isoforms andprotein misfolded-mediated disorders” WO 2004/070025]. The antibodiesN4, N7, N11, N14 and N27 also recognized an additional GPBP-relatedpolypeptide of −88-kDa, which may represent a phosphorylated version ofthe 77-kDa canonical polypeptide (Raya et al 1999 J. Biol. Chem. 274,12642-12649). The N26 antibody recognizes a 91-kDa polypeptide whichco-migrated with the recently characterized 91-kDa GPBP isoform (Revertet al. 2008 J. Biol. Chem. 283:30246-55) targeted by mAb e26 (FIG. 17).The relative efficiencies of the new monoclonal antibodies for detectionof GPBP isoforms (native or recombinant) have been estimated andsummarized in Table 2.

Epitope mapping for N1-N28 monoclonal antibodies. For these purposes, weproduced thirteen different cDNA constructs representing individualC-terminal deletion mutants of GPBP (FIG. 18A). The individualconstructs were used for HEK 293 cell transfection and the correspondingcell extracts analyzed by Western blot to assess individual antibodybinding. Seventeen out of the 28 new monoclonal antibodies recognizeddeletion mutant 8 but failed to recognize mutant 7 (Table 1); the restof the antibodies either target the N terminal end, or the epitope wasnot determined because of lack of reactivity in Western blot assays.Since the majority of the antibodies reacted with deletion mutant 8 andfailed to react with deletion mutant 7, we further attempted individualepitope mapping using synthetic peptides representing the sequencecomprised by between C terminal ends of deletion mutant 7 and 8.Strikingly, we failed confirming reactivity of the antibodies versusthese 40-residues and also these peptides could not compete GPBPantibody binding. Data suggested that existed a region that was highlyimmunogenic that required GPBP N terminal region for adequate epitopeassembly. This was investigated by producing FLAG-GPBP internal deletionmutants (Δ1-Δ4), in which only the indicated individual 20-residuesequences were removed (FIG. 18B). Deletion mutants Δ1-Δ4 were obtainedby standard procedures using two consecutive PCRs and specific primersto introduce the corresponding deletions (FIG. 18B). Interestingly, allthe antibodies failed to react with Δ2 and Δ3 internal deletionFLAG-GPBP mutants but reacted with Δ1 and Δ4 mutants (FIG. 18C). Dataindicate that the sequence represented by residues 305-344 of GPBP(GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIEEQ SQSEK, SEQ ID NO:10) conforms ahighly immunogenic epitope cluster. Consistently, previouslycharacterized mAb 14 was found to react with this region at the FATTmotif (Revert et al. 2008 J. Biol. Chem. 283:30246-55).Classification of the monoclonal antibodies. This has been performedtaking into consideration epitope mapping and reactivity with eithernative or recombinant intracellular or extracellular GPBP isoforms inWestern blot analysis (Table 1).

TABLE 1 Classification of the 28 monoclonal antibodies. Western blotreactivity with GPBP Mono- Recombinant clonal (77-kDa) Native (kDa)Region Group No. lysate medium 91 88 77 45 7-8  1a 4, 11, 14, 27 +++ +++− + +++ ++/+  1b 7, 22 +++ +++ − +₍₇₎/−₍₂₂₎ +++ − (~mAb14) 2 5, 10, 12,13, +++ ++/ − − ++_((5, 10, 12))/+ ++ 16, 25, 28 ±_((10, 15, 16)) 3 2,3, 8, 9 ++ ± − − +/± ±/− <7 4 15, 23 ++ + − − ± − <4 5 1, 18, 19, ++ −+₍₂₆₎ − − − 24, 26 ? 6 6, 17 +/± − − − − ±/− ? 7 20, 21 ±_((b))/− −Several polypeptides are targeted The numbers in the “Region” fieldrefer to the different deletion mutants used in the analysis (see FIG.18, upper composite). For example, Region 7-8 indicates that theantibodies recognize mutant 8 but not mutant 7, and Region <7 means theepitope is N terminal respect to the C terminus of mutant 7.Characterization of N1-N28 monoclonal antibodies by indirectimmunofluorescence analysis of HeLa cells expressing recombinant GPBP.HeLa cells were transfected with pcDNA3-FLAG-GPBP, cultured for 24additional hours, and fixed with methanol/acetone (50%-50%). Afterfixation, cells were blocked with 3% BSA in PBS (blocking solution) andincubated with the indicated antibodies (cultured media) diluted 1:2 inblocking solution. Subsequently, cells were washed with PBS andincubated with FITC-labeled anti-mouse IgG, washed again, mounted andobserved with an inverted fluorescence microscope. Images were acquiredwith a 40× objective using identical exposition times and gains. ExceptN6, all antibodies recognized FLAG-GPBP expressed in HeLa cells withdifferent reactivity, being the most reactive antibodies for thispurposes N13, N14, N15, N16, N21, N22 and N26 (see Table 2 for relativedetection efficiencies). Among reactive antibodies, all except N28unveiled the GPBP-characteristic reticular distribution pattern,consequence of the localization of GPBP at the endoplasmic reticulum(Revert et al. 2008 J. Biol. Chem. 283:30246-55).Characterization of N1-N28 monoclonal antibodies by indirectimmunofluorescence analysis of HeLa cells. HeLa cells were seeded andcultured on crystal slides, processed as above, and analyzed with aninverted fluorescence microscope using a 40× objective and an imageamplification of 1.63. Except N26, all antibodies showed endoplasmicreticulum distribution similar to that yielded by antibody N27 shown atthe left of the composite. Some cells showed also a peri-nuclear andfocal reinforcements typical of the Golgi apparatus (white arrow). Thepattern unveiled by N26 mixes the previously described reticulardistribution with nuclear and peri-nuclear punctuate clusters, andlinear decoration of plasma membrane. Except N26, all antibodiesexclusively unveiled the endoplasmic reticulum distribution describedfor recombinant GPBP polypeptide. Antibody N26, apart from yielding areticular pattern, decorated the plasma membrane and evidenced punctuateperi-nuclear and nuclear accumulations. The best antibodies fordetecting endogenous GPBP materials in HeLa cells were N5, N12, N16,N21, N26 and N27 (see Table 2).Characterization of N1-N28 monoclonal antibodies by immunohistochemicalanalysis of paraffin-embedded human kidney tissue. Individual monoclonalantibodies were used for standard immunohistochemical analysis ofparaffin-embedded human kidney samples. All the reactive antibodiesstained mainly convoluted and collecting tubules with significantstaining also within glomeruli at mesangial cells, podocytes, mesangialmatrix and capillary walls. In the later case with a linear pattern atthe endothelium surface and with a granular-like distribution within thecapillary wall. In capillary walls, immunostaining was less frequent,being N5, N6, N7, N8, N10 and N26 the best antibodies for thesepurposes. The antibodies rendering better GPBP detection usingimmunohistochemical techniques were N5, N6, N7, N8, N9, N10, N12, N26and N27 (see Table 2).Assessment of the ability of N1-N28 monoclonal antibodies to captureGPBP in a sandwich ELISA assay. In order to select individual antibodiesfor sandwich ELISA assays, an ELISA plate previously coated withanti-mouse antibody was used to bind monoclonal antibodies from culturemedia and their ability to capture recombinant and native GPBP assessed.Anti-mouse-coated ELISA plates were loaded with the culture medium fromthe hybridomas of the indicated antibodies or with the culture mediumfrom an anti-GAPDH hybridoma (cont). Subsequently, the plate was blockedwith 3%

BSA in PBS and incubated with recombinant GPBP diluted in FBS at theindicated concentrations, or with FBS (blank). Bound GPBP was detectedwith chicken polyclonal anti-GPBP and HRP-labelled anti-chicken IgY.Development was performed with a fluorescent reagent (Amplex).

-   -   a) Capture assays for human recombinant GPBP. All antibodies        efficiently captured FLAG-GPBP, with N5, N6, N8, N10, N11, N12,        N15, N16, N20, N23, N26, N27 and N28, displaying the best        efficiency capturing FLAG-GPBP from FBS containing 10 ng/ml        FLAG-GPBP (Table 2)    -   b) Capture assays for human circulating GPBP (plasma).        Anti-mouse-coated ELISA plates were loaded and blocked as above        indicated and further incubated with a Goodpasture patient human        plasma (register no. M049) diluted 1:10 in FBS or with FBS alone        (blank). Nine out of the 28 antibodies (N3, N5, N9, N10, N11,        N12, N13, N26 and N27) efficiently captured efficiently GPBP        from human plasma (Table 2).

CONCLUSION

We provide new monoclonal antibodies for native GPBP detection by ELISA,immunofluorescence and immunohistochemical procedures.

TABLE 2 Summary of the relative efficiency of the 28 monoclonalantibodies as detection antibodies in Western blot, immunofluorescence(IF) and immunohistochemistry (IHC), and as capture antibodies insandwich ELISA Western blot capture antibody recGPBP natGPBP (293) IF(sandwich ELISA) intracel extracel 45 77 88 91 rec nat IHC rec nat(serum) N1 ++ − − − − − + +/− +/− ++ − N2 +++ + − +/− − − +++ +/− +/−+/− − N3 +++ + − + − − ++ +/− + ++ + N4 ++++ ++++ + ++++ + − ++ +/− + +− N5 ++++ +++ ++ ++ − − ++ ++ ++ +++ ++ N6 + − − − − − +/− +/− ++ +++ −N7 ++++ ++++ − ++++ + − ++ +/− ++ + − N8 +++ ++ − +/− − − +++ +/− ++ +++/− N9 +++ ++ − +/− − − +++ +/− ++ ++ + N10 +++ + ++ +++ − − ++ +/− ++++ + N11 ++++ ++++ ++ ++++ ++ − ++ +/− − +++ ++ N12 +++ +++ +++ +++ − −++ ++ +++ +++ +++ N13 +++ +++ + + − − ++++ +/− + ++ ++ N14 ++++ ++++ +++++ ++ − +++ +/− + ++ − N15 +++ ++ − +/− − − +++ +/− +/− +++ − N16 ++++++ + ++ − − +++ ++ + +++ − N17 + − − − − − + +/− +/− +/− − N18 ++ − − −− − ++ +/− +/− +/− +/− N19 +++ − − − − − + +/− +/− +/− − N20 − − smear++ +/− + ++ +/− N21 − − smear +++ ++ + + − N22 ++++ ++++ − ++++ − − +++++/− +/− + − N23 ++++ + − − − − ++ +/− +/− ++ +/− N24 ++ − smear ++ + + ++/− N25 +++ ++ ++ + − − ++ +/− +/− + − N26 ++ − − − − ++ +++ ++ ++ ++ ++N27 ++++ ++++ ++ ++++ ++ − ++ ++ ++ +++ ++ N28 ++++ ++++ ++ + − − ++/− + ++ +/−

1. A method for detecting urinary Goodpasture antigen binding protein(GPBP), comprising (a) contacting a urine sample with an antibody thatbinds to 91 kD GPBP as set forth in SEQ ID NO:2 and/or with an antibodythat binds to 77 kD GPBP as set forth in SEQ ID NO:4, under conditionsto promote selective binding of the antibody to the 91 kD GPBP and/or tothe 77 kD GPBP; (b) removing unbound urine and/or antibodies; and (c)detecting complex formation between the antibody and the 91 kD GPBP,and/or between the antibody and the 77 kD GPBP in the urine sample. 2.The method of claim 1, wherein the method comprises detecting native 91kD GPBP from human urine.
 3. The method of claim 1, wherein the methodcomprises detecting native 77 kD GPBP from human urine.
 4. The method ofclaim 1, wherein the antibody is a monoclonal antibody.
 5. The method ofclaim 1, wherein the detecting comprises a technique selected from thegroup consisting of ELISA, immunoflourescence, flow cytometry, andchromatography.
 6. The method of claim 1 wherein the urine sample isobtained from a human subject suspected of having a disorder selectedfrom the group consisting of Goodpasture Syndrome and immune-complexmediated glomerulonephritis.